Antimicrobial Effect of Nano-Silver Against Oral Streptococci: Implications in Containment of Bacterial Biofilm on Orthodontal Appliances

Among various metal-based nanoparticles, silver nanoparticles (AgNPs) manifest superior inhibitory effect against several microorganisms. In fact, the AgNPs based therapy has been reported to inhibit both sensitive as well resistant isolates of bacteria and other disease causing microbes with equal propensity. Keeping this fact into consideration, we executed bio-mediated synthesis of AgNPs employing Hibiscus rosa sinensis flower extract. The as-synthesized AgNPs were evaluated for their potential to inhibit Streptococcus mutans (S. mutans), one of the main causative bacteria for dental caries. Beside several other reasons, orthodontic appliances have also been reported to facilitate infliction of oral cavity with a range of microbes including S. mutans. To determine the growth inhibitory and anti-adherence activity of AgNPs on orthodontic appliance, we performed microbiological assays employing AgNPs adsorbed on to the orthodontic wires. Topographic analysis of orthodontic wires was executed by scanning electron microscopy. In addition to antimicrobial and antibiofilm activity against oral S. mutans, the as-fabricated AgNPs demonstrated significant inhibitory and anti-biofilm properties against other biofilm forming bacteria such as Escherichia coli and Listeria monocytogenes as well.


Introduction
The nano-sized metal particles exhibit altered chemical and physical attributes as compared to their precursor chemical entities. Considering this fact, there has been an increasing trend in exploitation of metal-based nano-particles in diversified fields ranging from optics, catalysis as well as in medical diagnostics and therapeutics etc [1][2][3]. Well-dispersed and ultrafine metal nano-particles acquire distinctive thermodynamic properties when compared to their precursor metal compounds. Because of their nano order size dimensions, high energy surface atoms, greater penetration and ability to target various vital biological molecules of the host, the nano-particles have been widely used in various medical science related fields as well [2,3].
It is noteworthy that the nanometer size of the particles renders them the ability to traverse various biological barriers of the recipient's body and even guide them to accumulate in tissues either beneath the skin or deeper into the organs like lungs, liver and even less accessible component of CNS such as brain [4]. As a consequence, the NP based drug delivery systems have been widely exploited in treatment of cancer and several infectious diseases as well [5].
Various chemical and physical methods such as UV or microwave irradiation, chemical reduction, photochemical process, electron irradiation etc., have been widely used in the synthesis of nano-particles [6]. Most of these methods involve multistep processing, requirement of high energy input or involvement of hazardous chemicals to carry on NP synthesis. Moreover, most of the above-mentioned methods require relatively sophisticated instrument for NP synthesis and also suffer from the limitations such as low material conversion and difficulty in purification etc. On a positive note, there has been a sudden shift in the focus of scientific community towards exploitation of plants and their extracts as reducing cum stabilizing agents and their usage in the synthesis of metallic nano-particles.
The 'green synthesis' approach has been receiving increased attention as it is environment friendly and causes almost negligible pollution of any kind [7]. Further, the cost effective green synthesis technique has edge over other synthesis methods and can be easily modulated for large scale production [8]. Moreover, by smart maneuvering of fabrication conditions, green method can be manipulated to synthesize nano-particles with diverse shapes and range of sizes [7,8]. 4 Dental caries, a multi-factorial disease, has been considered as a serious oral health issue. It is more common in individuals, who wear orthodontic appliances. The orthodontic devices not only offer a platform to growing microbes, however, pose an obstacle in biofilm removal during oral hygiene procedures. The disease leads to demineralization and erosion of the tooth enamel surface. Prolonged dental plaque facilitates accumulation of Streptococcus mutans (S. mutans), the causative oral pathogen, that in turn ensues in development of dental caries [9]. Moreover, various orthodontic elastomeric modules, including brackets and wires, when accompanied with poor oral hygiene may also lead to establishment of infection caused by various oral bacteria such as S. mutans [10]. The bacterial infection is generally accompanied with formation of biofilm on both tooth enamel as well as orthodontic appliances used in dental care setup [11]. The related oral clinical issues may result in unacceptable esthetic presentation. If not treated properly, the infection not only causes bacteremia, however, also accompanied with inflammatory response in the afflicted patients.
The infection may lead to infective endocarditis as well [12]. Among various metal-based nano-particles, silver nano-particles possess excellent antimicrobial property against wide range of microorganisms. The special treatment of orthodontic appliances with NPs, before their usage in clinical settings, is likely to combat invading pathogens by manifesting strong anti-biofilm activity [13].
Hibiscus rosa-sinensis Linn (Family Malvaceae) is reported to possess potent medicinal properties including antitumor, cardio-protective, hypotensive, antidiabetic, anticonvulsant wound and antioxidant activity [14]. The Hibiscus rosa-sinensis flowers (HRSF) are known to contain flavonoids like anthocyanin and quercetin. The HRSF flavonoids have also been implicated for their anti-depressant and anxiolytic activity [15].
In the present study, we first ascertained the potential of a novel HRSF extract to synthesize silver nano-particles. The characterization of as-synthesized silver nano-particles (AgNPs) revealed that in a manner similar to other plant or bacterial exudates, HRSF extract too has potential to facilitate fabrication of nanosized particles in a time and concentration dependent manner. The kinetics of as-synthesized AgNPs synthesis was monitored employing UV spectrophotometry. The resultant NPs were characterized using various spectroscopic techniques. The antibacterial and anti-biofilm potential of as-formed AgNPs was evaluated against range of bacteria including caries causing S. mutans. Further, we also assessed antiadherence potential of AgNPs against S. mutans adsorbed on orthodontic wires. 5

Hibiscus rosa-sinensis flower extract preparation. Fresh flowers of Hibiscus rosa-sinensis
were collected and washed several times with deionised water. A known volume of distilled water (100 ml) was mixed with 10g (wet weight) of thoroughly washed HRS flowers. The mixture was boiled till the original volume of aqueous suspension reduced to 50ml and filtered through a Whatman No. 1 filter paper. The resulting HRSF extract was used in synthesis of as-fabricated AgNPs. UV absorbance spectroscopic studies. UV spectroscopic analysis of the as-synthesized nanoparticles was done to ascertain biogenic reduction of precursor silver nitrate salt. The double beam spectrophotometer (Jasco model V-750), operated at a resolution of 1 nm, was used to execute analysis (8). The UV-VIS spectra was recorded over the 200-800 nm range.

Fourier transform infrared spectroscopy (FT-IR) measurement. FT-IR of as-formed
AgNPs was analyzed on Perkin-Elmer FT-IR spectrophotometer in the diffuse reflectance mode operating at a resolution of 4 cm -1 (9). The samples were run in triplicate to validate the readings. The spectra were taken between 4000 cm -1 and 500 cm -1 by averaging 128 cm scans for each spectrum. monocytogenes. They were grown overnight to attain the colony-forming unit (CFU) counts of ∼10 6 units per ml. An aliquot of 100µl, from overnight grown culture, was spread uniformly on nutrient agar petri-plate by sterile plastic spreader.
The agar plate, inoculated with S. mutans, was co-cultured with AgNPs Ni-Ti orthodontic wire. The uncoated as well as plain AgNO3 coated Ni-Ti orthodontic wires were used as control. The plates were incubated at 37 °C and the zone of inhibition was observed after the stipulated time period of 24hrs.
In the next set of experiment, the agar plates were first exposed with E. coli and L. monocytogenes followed by boring of the wells using punching machine. Subsequently, an increasing amount of as-synthesized AgNPs (1 mg/ml stock solution) or standard antibiotic, gentamicin (control) was dispensed into the various wells in a given agar plate. The plates were incubated at 37 °C and the zone of inhibition was determined by measuring the clear region around each well after the stipulated time period of 24hrs.

Antibiofilm potential of as-synthesized AgNPs employing XTT assay
The anti-biofilm potential of as-synthesized AgNPs was established employing XTT assay following published protocols with minor changes as standardized in our lab. Briefly, 100 µl of bacterial cell culture (1x10 8 cells/ml) was dispensed in each well of 96 well culture plates.
The bacteria were allowed to form adherent mature biofilm in the presence of increasing concentration of the as-formed AgNPs for 48hrs. Just before the commencement of the assay, a solution of Menadione (0.4 mM) was prepared and filtered.
The plate was incubated for stipulated time period, followed by washing with PBS (200ul).
-2H-tetrazolium hydroxide (XTT) at a final concentration of 250 µg/ml] was dispensed to each test well of the plate followed by addition of menadione solution (2 μl/well). The plate was further incubated for the next 4hrs at 37 °C in the dark. Next, the solution was transferred into a fresh plate to assess the colorimetric change using a microtitre plate reader (BIORAD Microplate reader at 490 nm). The XTT experiments were performed in triplicate to validate the data.

Effect of as-synthesized AgNPs on the adherence of S. mutans to Smooth Glass Surfaces in presence of sucrose
We studied effect of as-synthesized AgNPs on the adherence of S. mutans on smooth glass surface. We also assessed effect of presence of sucrose in the surrounding medium during AgNP mediated inhibition of S. mutans adherence. The bacteria were cultured at 37 °C for 24 h at an angle of 30° in a glass tube containing 10 ml of BHI with or without 5% (w/v) sucrose. The cultured tubes were also exposed with the sub-MIC concentrations of the AgNPs. The culture medium was consisting of BHI with (sucrose dependent) and without sucrose (sucrose independent). After stipulated incubation, the glass tubes were slightly

Hydrophobicity index of as-synthesized AgNPs treated bacterial biofilm
Overnight cultured bacterial cells (treated and control) were suspended in 1 ml LB medium to obtain optical density (at wavelength of 595nm) at around 1.0 ± 0.01. Further, toluene (1ml) was added to the suspension (both treated and control) and vortexed. The test tubes were incubated for 30 mins to let the biphasic solution to settle. Next, the optical density of the aqueous phase was measured and the hydrophobicity index was calculated by using the equation: Where Ai and Af denote the initial and final optical densities of aqueous phase. The adherence of the bacterial cells to the organic solvent was evaluated to ascertain the hydrophobicity [16].

Anti-adherence properties of as-synthesized AgNPs
Sterilized Ni-Ti orthodontic wires were placed into three separate Eppendorf tubes, having fresh sterilized nutrient broth. Next, one of the two sets of orthodontic wires was co- Anti-Biofilm activity as revealed by fluorescence microscopy.
S. mutans biofilm, cultured on sterile glass cover slip, was placed in the well of multi-well polystyrene plate. Briefly, S. mutans cell suspension (at the 1.0 absorbance O.D600 in NB medium) was cultures on a coverslip overnight at 37°C. After 24h incubation, media was replenished and biofilm was exposed to AgNPs suspension for duration of 3 hrs. Untreated mature biofilm was considered as control. After treatment with AgNPs and aqueous AgNO3solution, the plate was washed with PBS, and the anti-biofilm activity was observed by fluorescence microscopy.

Quantification of extracellular glucan in cultures with sucrose
The phenol/H2SO4 method was used to quantify the glucan in the cultured sample. S. mutans was incubated in media containing 1% sucrose both in absence and presence of assynthesized AgNPs for 20 h, after which the bacteria were recovered. Extracellular watersoluble glucan was isolated by employing ethanol-precipitation of the supernatants from the recovered bacterial cultures. The remaining cell pellet was re-suspended in 1 M NaOH and centrifuged to remove bacterial cells. Water-insoluble glucan was recovered by ethanolprecipitation of the supernatant.

Assessment of topographical changes in orthodontic wires
Ni-Ti orthodontic wires were incubated with as-synthesized AgNPs and aqueous AgNO3 for 24 hrs with mild and continuous shaking for homogenous adherence. Topographical changes on coated Ni-Ti orthodontic wires as compared to uncoated one (control) were assessed by Scanning Electron Microscopy.

Hibiscus flower extract mediated synthesis of metal nanoparticles
The potential of HRSF extract to synthesize nano particles was established by evaluating its ability to convert silver salt to as-synthesized silver nanoparticles. The aqueous silver ions were exposed to the HRS flower extract. The color of the reaction mixture changed from pale yellow to blackish grey. The color transformation corresponds to characteristic surface plasmon resonance (SPR) effect of different sized silver nanoparticles. SPR analysis establishes HRSF mediated synthesis of Ag NPs.

UV-VIS spectra of silver nanoparticles
The optical properties of the as-formed AgNPs were determined employing surface plasmon absorption spectroscopy. The SPR properties usually depend on the shape of NP population.
At the outset, a UV-visible absorption spectroscopic technique was used to analyze the synthesis of the nanoparticles. Figure 1A shows the UV-VIS absorption spectra recorded for as-synthesized silver nanoparticles harvested after 24 hrs of reaction (upon treatment with 3ml of HRSF extract). The results indicate that the reaction solution displays an absorption maximum at about 480 nm that can be attributed to the SPR of the as-formed silvernanoparticles [17]. It was observed that with increase in time, silver nano-particle synthesis rate also increased (data not shown). Although, the wavelength maximum was stably positioned at 480 nm, however, the intensity steadily increased with the time and eventually got saturated after 24 hrs. The longitudinal plasmon exhibited a change in position with time (data not shown).
We also studied the kinetics of silver nanoparticle synthesis upon incubation of AgN03 with increasing content of HRS flower extract. The synthesis of AgNPs augmented upon increasing the concentration of plant extract in the reaction medium. The increase in HRSF content (3ml vs 5ml HRSF extract) resulted in upsurge in absorbance at 480 nm ( Figure 1A).

TEM and DLS Analysis
The biomimetic synthesis of silver nano-particles was further confirmed by representative TEM images of silver nanoparticles generated employing increasing amounts of flower extract for their synthesis. The shape of the particles was found to be dependent on the concentration of flower extract. Higher abundance of HRSF extract favored the occurrence of large homogenous population of small sized nano particles. At lower concentration of flower extract various triangular and hexagonal and ovoid shape particles in the size range 10-40 nm ( Fig 1B) were seen to be coexisting. However, as the concentration of HRSF extract was increased, the average diameter of the silver nanoparticles was found to decrease as evidenced by the occurrence of spherical particles (15-25nm) at higher concentration of HRS flower extract ( Figure 1C). Also, as the flower extract concentration increased, the total number of particles in a given volume increased simultaneously as seen in the TEM micrograph ( Figure 1C). Initially, the increase in the HRSF extract causes the nanoparticles to be formed in the range of 30-50 nm. This is more evident from the fact that higher

Surface Charge of AgNPs as measured by Zeta Potential
Zeta potential is a physical property which relates to the net surface charge of the nanoparticles. Depending upon the magnitude and nature of the charge, the particles may repel each other (because of the Coulomb explosion force) that eventually prevent agglomeration of the as-formed nanoparticles. The stability of NPs has been correlated well with the zeta potential ranging from +30 mV to -30 mV. The as-synthesized AgNPs were found to have zeta potential around-25 mV [ Figure 1E].  Figure 2].

Anti-bacterial potential of AgNPs as assessed by agar well diffusion method
The antimicrobial activity of AgNPs was assessed against S. mutans, using the agar well diffusion assay. The zones of inhibition (in mm) around each well containing AgNPs had been assessed (Figure 3). We also determined antibacterial activity of the as-synthesized AgNPs against E. coli and L. monocytogenes. The AgNPs exhibited significant antibacterial activity against all the tested organisms. Besides inhibiting cell-to-cell interaction, the AgNPs also induced ROS production. This led to fragmentation of DNA. The antibacterial activity of AgNPs can also be attributed to the inhibition and/or disruption of respiratory chain and cell division process that ultimately leads to cell death [18]. The silver nanoparticles have been reported to release silver ions inside the bacterial cells thereby inciting killing of the treated bacteria [19].
Ni-Ti orthodontic wires were coated with as-synthesized AgNPs or AgNO3 solution. The impregnated wires were placed on the agar plate that was previously inoculated with resistant isolates of S. mutans. The wire impregnated with AgNPs showed significant S. mutans inhibition (clear zone of inhibition) as compared to AgNO3 coated wire (Figure 4).

Anti-adherence potential of as-formed AgNPs
Ni-Ti orthodontic wires (coated with AgNPs or AgNO3) were placed into Eppendorf tubes.
The wire harbouring tubes were co-exposed with fresh NB broth along with suspension of less sensitive S. mutans. On completion of stipulated incubation period, wires were taken out and placed into fresh Eppendorf tubes containing PBS and subjected to sonication to remove adhered bacteria. The detached bacteria were cultured on agar plate. The wire treated with AgNPs showed less bacterial load as compared the one that was treated with aqueous AgNO3 solution (Figure 5a and 5b).

Anti-biofilm potential of as-synthesized AgNPs employing XTT assay
The as-synthesized AgNPs inhibited adherence of S. mutans to glass tubes. The assynthesized AgNPs inhibited both sucrose-independent as well as sucrose-dependent adherence of the treated S. mutans bacteria. The inhibition was more pronounced in sucrosedependent adherence as compared to the treated sucrose free medium. The as-synthesized AgNPs inhibited the S. mutans biofilm formation in a dose-dependent manner ( Figure 6A).
The AgNPs at a concentration of around 6.25 g/ml inhibited around 50% of the formed biofilm.

AgNPs mediated hydrophobicity inhibition of the treated Streptococcus mutans
The as-synthesized AgNPs were found to inhibit cell surface hydrophobicity of the S. mutans in a concentration-dependent manner as shown in figure 6B. The As-synthesized AgNPs reduced the hydrophobicity to more than 50% at a concentration of ~100 µg/ml.
We also determined anti-biofilm potential of as-synthesized AgNPs against less susceptible isolates of L. monocytogenes and E. coli. Increasing amount of AgNPs (ranging from1.56 to 200μg/ml) was dispensed in each well of microtiter plate. Subsequently, the plate was inoculated with a bacterial strain (for 24 h) as specified in method section. It can be inferred from the results that the biologically synthesized AgNPs based formulation was successful in inhibiting the drug resistant bacterial biofilm (less susceptible against gentamycin), as compared to the negative control ( Figure S2). The adherence potential of bacterial cells can be attributed to the high hydrophobicity associated with the bacterial cell surface. The percentage of hydrophobicity index for E. coli (less susceptible isolate) decreased from ~58% to ~34% on treatment with as-synthesized AgNPs. Similarly, the hydrophobicity index for less susceptible L. monocytogenes isolate decreased from ~64 % to ~ 43 % ( Figure S2).

Anti-Biofilm activity as revealed by fluorescence microscopy
To ascertain the anti-biofilm potential of the as-formed AgNPs, we assessed S. mutans (less susceptible isolate) mediated synthesis of biofilm on glass surface. The fluorescence microscopy reveals formation ofmature biofilm when S. mutans was cultured in absence of AgNPs. It could be attributed to unhindered proliferation of untreated cells leading to biofilm formation. On the other hand, the treatment with AgNPs resulted in the inhibition of biofilm formation on the glass surface [ Figure 7].

The as-synthesized AgNPs inhibited glucan synthesis in the treated S. mutans cells
We determined potential of the as-synthesized AgNPs to inhibit glucan synthesis in the treated S. mutans. The AgNPs efficiently inhibited glucan synthesis in a concentrationdependent manner. The AgNPs inhibited around 50% glucan synthesis at around 100 µg /ml concentration ( Figure 6).

Assessment of topographical changes in orthodontic wires
Surface morphology or the topographic conditions of Ni-Ti orthodontic wire was studied by SEM analysis (Figure 8             Hydrophobicity assay. As-synthesized AgNPs were able to inhibit glucan synthesis by S. 19 mutans(C) as well as also resulted in its decreased adherence potential on glass surface (D).
[*: P value < 0.001, **: P value < 0.005]   leading to peri-implantitis that is considered to be the main reason for implant failure.
Interestingly, metal-based nano-particles offer a salvage strategy to treat oral infection. In this regard, silver based nanoparticles (AgNPs) can successfully control infection [18]. Moreover, synthesis of nanoparticles employing biological methods i.e. green synthesis offers additional benefits in terms of biocompatibility and bioavailability. Hence, we explored HRFS based extract for its potential to synthesize AgNPs. Next, we examined the antibacterial and antiadherence activity of as-synthesized AgNPs on the surface of Ni-Ti orthodontic wires against S. mutans.
To begin with, first we characterized as-synthesized AgNPs for their physico-chemical attributes. To track the crystallization progress, TEM and UV spectro-photometric studies were carried out. The HRSF mediated biological synthesis lead to formation of spherical AgNPs, with size distribution in the range of 15-40 nms. The DLS analysis suggested the size dimension of as-synthesized AgNPs in the range of 30-80 nm. The discrepancy in the observed average size of the as-synthesized particles can be attributed to the method used in the size determination (19).
The as-synthesized AgNPs were found to exhibit characteristic SPR showing absorption peak in the range of 480nm. In accordance with Mie theory, a single SPR band is obtained in case of spherical nanoparticles. The FT-IR spectroscopy also suggested formation of a new microambience corresponding to functional groups of the parent compounds present in HRS flower extract.
The zeta potential values ranging from +30 to -30 mV are supposed to help in maintaining the stability of the related nanoparticles [20]. Incidentally the as-synthesized AgNPs were found to have zeta potential around -35 mV. This suggests that HRSF extract mediate synthesis of stable AgNPs.
Keeping into consideration usefulness of silver nanoparticles in treatment of range of microbes, we tested their anti-biofilm potential against various other classes of bacteria in addition to S. mutans. We made an elaborated effort to ascertain the effect of as-synthesized AgNPs against both resistant as well sensitive bacterial isolates. The anti-biofilm activity, as revealed by XTT assay, suggests that as-synthesized AgNPs can significantly inhibit less susceptible isolates of S. mutans, E. coli and L. monocytogenes. The observed effectiveness of as-synthesized AgNPs to kill both sensitive as well as less susceptible isolates of bacteria has great relevance in the present scenario where drug resistance crisis by microbes is posing great health concern. Moreover, as-synthesized AgNPs were also shown to inhibit glucan synthesis by S. mutans.
We established potential of Ag NPs to inhibit bacterial growth on orthodontic appliances.
The AgNPs deposited onto the surface of experimental dental appliances including orthodontic wire showed strong antimicrobial properties. In general, orthodontic appliances serve as a favorable site for S. mutans to adhere, by forming a bacterial biofilm on their surface. However, if coated with AgNPs, they showed significant anti-adherence properties as well. The electron microscopic studies further established anti-adherence activity of the AgNPs on orthodontic wires (Figure 8).
While practice of establishing antimicrobial activity of AgNPs against various pathogens is a common feature, the studies pertaining to anti-adherence potential of AgNPs in context of orthodontic wires are limited [21]. This makes the present approach more relevant as we have demonstrated that coating of AgNPs on to the surface of orthodontic wire impart antiadherence properties to the later. This observation is very interesting and has a great relevance as orthodontic wire and other related dental appliances are very prone to be contaminated by various oral bacteria including S. mutans.
Earlier studies have suggested that AgNPs have the ability to release silver ions that have potential to kill target bacterial cells [22]. The observed anti-adherence property of the AgNPs may be attributed to the fact that they can penetrate the cell wall of associated bacteria and also prevent production of extracellular polysaccharide to check bacterial adhesion on orthodontic wire.
The immersion of orthodontic wire in AgNPs suspension followed by their interaction with S. mutans on the agar plate ensued in killing (clear zone of inhibition) of bacteria present in the surrounding ambiance. There is a possibility that AgNPs may interact with thiol or amino residues of various crucial enzymes and eventually lead to inhibition of the bacterial cells [23]. The large surface area of as-formed AgNPs is likely to facilitate inhibition of various metabolically active enzymes. Moreover, AgNPs have also been reported to facilitate generation of reactive oxygen species under oxygen atmosphere [24]. The potential of asgenerated ROS to act on proteins and DNA based macromolecules has been considered detrimental to the live bacterial cells [25]. The higher adhesion of S. mutans can be attributed to the surface morphology of the uncoated (bare) orthodontic wires [ Figure 4]. The rougher surfaces are known to encourage biofilm formation and support greater adherence. In contrast, the treatment with AgNPs minimizes irregular cavities and undulating surface of orthodontic wires, thereby minimizes adherence of S. mutans (Figures 4 & 8).
Finally, we can conclude that as-formed AgNPs showed remarkable antibacterial potential against both resistant as well as sensitive isolates of the bacteria. The adsorption of assynthesized AgNPs on orthodontic wire imparts strong antibacterial attributes to the Ni-Ti orthodontic wires. We speculate that AgNP impregnated, orthodontic wires acquire intrinsic potential to inhibit bacterial growth upon their use in clinical setting.