Titanium Dioxide Nanoparticles Induce Inhibitory Effects against Planktonic Cells and Biofilms of Human Oral Cavity Isolates of Rothia mucilaginosa, Georgenia sp. and Staphylococcus saprophyticus

Multi-drug resistant (MDR) bacterial cells embedded in biofilm matrices can lead to the development of chronic cariogenesis. Here, we isolated and identified three Gram-positive MDR oral cocci, (1) SJM-04, (2) SJM-38, and (3) SJM-65, and characterized them morphologically, biochemically, and by 16S rRNA gene-based phylogenetic analysis as Georgenia sp., Staphylococcus saprophyticus, and Rothia mucilaginosa, respectively. These three oral isolates exhibited antibiotic-resistance against nalidixic acid, tetracycline, cefuroxime, methicillin, and ceftazidime. Furthermore, these Gram positive MDR oral cocci showed significant (p < 0.05) variations in their biofilm forming ability under different physicochemical conditions, that is, at temperatures of 28, 30, and 42 °C, pH of 6.4, 7.4, and 8.4, and NaCl concentrations from 200 to 1000 µg/mL. Exposure of oral isolates to TiO2NPs (14.7 nm) significantly (p < 0.05) reduced planktonic cell viability and biofilm formation in a concentration-dependent manner, which was confirmed by observing biofilm architecture by scanning electron microscopy (SEM) and optical microscopy. Overall, these results have important implications for the use of tetragonal anatase phase TiO2NPs (size range 5–25 nm, crystalline size 13.7 nm, and spherical shape) as an oral antibiofilm agent against Gram positive cocci infections. We suggest that TiO2NPs pave the way for further applications in oral mouthwash formulations and antibiofilm dental coatings.


Introduction
The enormously rich and complex salivary environment of the human oral cavity provides a uniquely structured habitat for a wide variety of commensal (aerobic/anaerobic) microorganisms, and more than an estimated 700 species [1] colonize the oral cavity and form biofilms to ensure their long-term survival. Moreover, notorious biofilm persisters, including streptococci and lactobacilli, live as mutual symbionts within biofilms [2]. Furthermore, it has often been speculated that oral microbiota (bacteria, yeasts, and viruses) promote biofilm formation by producing heterogeneous extracellular polymeric substances (EPS), proteins, and nucleic acids [3]. According to Tawakoli et al. [4], the most dominant oral diseases (caries and periodontitis) are caused by bacterial adherence and subsequent biofilm formation. Multi-layered bacterial biofilm matrices play a vital role in neutralizing the antimicrobial effects of various chemical agents by acquiring drug resistance

Isolation and Culture Conditions
The Gram positive, oral coccoid strains, SJM-04, SJM-38, and SJM-65 were isolated from the Outpatient Department (OPD) of the Periodontics and Community Dentistry Clinic, Dr. Ziauddin Ahmad Dental College and Hospital, Aligarh Muslim University, India, by swab sampling as described by Papaioannou et al. [20]. In detail, sterile pure viscose swabs (PW043, Hi-media, Mumbai, India) were used to collect saliva samples from the floor, subgingival, and gingivae of the buccal cavities of patients. Swabs were then immediately immersed into 10 mL of sterile normal saline solution (NSS) for 30 min. Subsequently, 1000 µL samples were spread onto brain heart infusion (BHI) ( were isolated based on phenotypic characteristics (shape, size, color, margin, and colony elevation), purified, cultured, and preserved/stored in 20% glycerol at −80 • C.

Assessment of Biofilm Formation at Different pH Values, Temperatures, and Salinities
Biofilm formation by the three isolates was assessed at different temperatures, pH values, and salinities. Georgenia sp. (SJM-04), S. saprophyticus (SJM-38), or R. mucilaginosa (SJM-65) were exposed to these various conditions in 96-wells microtiter plates. To assess the effect of pH stress, BHI broth was adjusted to pH 6.4, 7.4, or 8.4 with 0.1 M HCl or 0.1 M NaOH. For salinity tolerance, the concentration of sodium chloride (NaCl) was increased from 200 to 1000 µg/mL, and to assess the effects of temperature, microtiter plates containing pristine BHI medium were subjected to 28 • C, 37 • C, or 42 • C. Wells were inoculated with 20 µL of freshly grown test strains ( ∼ =1 × 10 6 /mL) in BHI broth. All experiments were performed in triplicate using independent bacterial colonies and data were averaged.

Phylogenetic Characterization of Oral Bacteria
The 16S rRNA gene amplicons of the three Gram positive oral isolates were amplified by PCR using the primers: 16S-27F (5 to 3 AGAGTTTGATCMTGGCTCAG, M = A or C) and 16S-1492R (5 to 3 ACGGCTACCTTGTTACGA) (Sigma-Aldrich, St. Louis, MO, USA). Qiagen DNeasy kits (Valencia, CA, USA) were used for genomic DNA extraction. For polymerase chain reaction (PCR) amplification, reaction mixtures containing 2.5U Taq polymerase (Sigma Aldrich), 100 µM of each dNTP, 0.2 µM of each primer, and 3 µL of DNA template (substrate for Taq DNA polymerase) in 50 µL of 2 mM MgCl 2 solution were processed using a thermal cycler and the following program: 96 • C for 2 min (denaturation), followed by 30 amplification cycles of 95 • C for 15 s, 49 • C for 30 s, and 72 • C for 1 min, and a final extension at 72 • C for 1 min. PCR products were purified using the QIAquick-spin PCR Purification Kit (Qiagen, Chatsworth, CA, USA) and sequenced in a DNA sequencing facility using the BioEdit sequence alignment editor. Gene sequence homology was determined using archived 16S rRNA sequences in the GenBank server (www.ncbi.nlm.nih.gov/nucleotide) accessed on 24 January 2019, BLAST Multiple alignments of sequences, and Clustal W program. Phylogenetic trees were constructed using MEGA 6.0 and the neighbor-joining (NJ) DNA distance algorithm with bootstrap analysis (1000 replications).

Physicochemical Characterization of TiO 2 NPs
The physicochemical characteristics of TiO 2 NPs (Sigma-Aldrich, St. Louis, USA; product code 637254) were determined using: (i) a double beam UV-Visible spectrophotometer (UV 5704S from Electronics, India, Ltd., Panchkula, India), (ii) an X-ray diffractometer, (XRD, Rigaku Corporation, Tokyo, Japan), (iii) a transmission electron microscope, (iv) a scanning electron microscope (JSM 6510LV, SEM, Tokyo, Japan), and (v) by energydispersive X-ray (EDX) analyses (Oxford Instruments INCAx-sight EDX spectrometer, Concord, MA, USA). Details of the material characterization methods are provided in our earlier study [22]. Average TiO 2 NP crystalline size was determined using the Debye-Scherrer's formula (D = 0.9λ/βcos θ; where D is crystal size, λ is X-ray wavelength, and β is the full-width at half-maximum (FWHM) of the diffraction peak). The dose-dependent antibacterial effects of TiO 2 NPs on isolated strains were determined. First, 100 µL of freshly grown (OD 600 = 0.01) SJM-04, SJM-38, or SJM-65 strains were added to microtiter wells containing 200 µL of BHI-TiO 2 NPs (250, 500, 1000, or 2000 µg/mL) suspensions, and incubated at 37 • C in triplicate for 24 h. Untreated and treated bacterial cells were then diluted by a factor of 10 −4 (OD 600 = 0.01) with sterile distilled water. To determine viable cell counts, 100 µL of diluted samples (OD 600 = 0.01) were spread on BHI agar plates and incubated at 37 • C for 24 h. The viabilities of test strains were determined by comparing the total plate counts (TPCs) of treated and untreated cells. Cells treated with or without TiO 2 NPs (250 µg/mL) were also examined for TiO 2 NP-induced morphological damage by SEM. Briefly, untreated and treated bacterial cells were spun at 3000 rpm for 5 min, fixed in glutaraldehyde (2.5%) at 4 • C for 4 h, and cell pellets were dehydrated in an ethanol series (30,50,70, and 90% ethanol for 15 min/step). A sample (100 µL) from each strain was mounted on a clean glass coverslip and coated with a thin layer of gold. Finally, the samples were examined under an SEM at 15 kV and 3000× [23].

Dose-Dependent Effect of TiO 2 NPs on Biofilm Formation
The dose-dependent effects of TiO 2 NPs on biofilm formation by the three isolates were quantified by measuring crystal violet (CV) absorbance, as described by Ahmed et al. [24]. In detail, 100µL ( ∼ =1 × 10 7 cells) of freshly grown SJM-04, SJM-38, or SJM-65 cells were added to wells containing 250, 500, 1000, or 2000 µg/mL of TiO 2 NPs in 200 µL of BHI broth per well. Cultures grown without TiO 2 NPs and sterile BHI broth alone were used as positive and negative controls, respectively. Micro-well plates were incubated at 37 • C for 24 h, and then TiO 2 NPs-BHI suspensions and loosely attached bacteria were carefully removed from the wells. Adherent biofilms on micro-well surfaces were then incubated with 200 µL of CV (1%) for 30 min, were washed with sterile PBS to remove nonabsorbed CV, and air-dried. Biofilm bound CV was then solubilized with ethanol (95%) and absorbances (OD 620 ) were measured using a microplate reader (Thermo Scientific Multiskan EX, REF 51118170, Shanghai, China). In a similar manner, biofilms were formed in 96-well plate for 24 h and these mature biofilms were treated with TiO 2 NPs to check the dispersal of mature biofilms by CV assay. In addition, TiO 2 NP-induced reductions in biofilm formation were also assessed by microscopy as described by Ahmed et al. [25]. Briefly, using the same conditions mentioned above, biofilms adherent to glass coverslips were washed with PBS to remove loosely attached planktonic cells and then stained with CV (1%) for 30 min. Air-dried biofilms on cover glasses were examined under an optical microscope (Olympus BX60, Model BX60F5, Olympus Optical Co. Ltd. Tokyo, Japan) equipped with a digital camera (Sony, Model no. SSC-DC-58AP, Tokyo, Japan).

Statistical Analyses
Multiple comparisons versus controls were performed by one-way analysis of variance (ANOVA) using the Holm-Sidak method (Sigma Plot ver. 11.0, San Jose, CA, USA). Results are presented as the means ± SDs of at least two independent experiments performed in triplicate. Statistical significance was accepted for p values < 0.05.

Isolation and Characterization of Oral Bacteria
The diverse oral microbiota within biofilms obtain the proteins and glycoproteins (mucins) they require to thrive from saliva [26], which is produced at a rate of 1.5-2.0 mL/min [27] and normally supports bacterial proliferation of ∼ =10 9 cells/mL [28]. Therefore, we collected human saliva with sterile swabs and subsequently added sterile saline solution enriched to near-physiological saline conditions, i.e., to millimolar concentrations of NaCl and Ca 2+ ions. Figure 1 shows the primary characteristics of the colonies of oral isolates, such as color, elevation, margin ( Figure 1(AI-AIII,BI-BIII)), morphologies, and  (Table 1). Resistance against thirdgeneration cephalosporin reflects the presence of single-nucleotide polymorphisms (SNPs) that directly increase CAZ hydrolysis by highly conserved class A β-lactamase [29] bacterial isolates. Similarly, Higashida et al. [30] in a study on eight S. saprophyticus strains also showed β-lactam resistance was due to mecA gene-mediated resistance. Moreover, transposon mutagenesis experiments have confirmed the role of mecA in conferring methicillin resistance [31]. Besides presenting as urinary tract infection bacterium, S. saprophyticus has been isolated from a variety of other samples such as different brands of minas cheese and beach water [32].

Biochemical Characterizations of the Three Oral Isolates
The survival of oral communities largely relies on the nature of the salivary environment (pH, temperature, and ionic strength) and the intrinsic metabolic responses of these communities to the salivary biochemical milieu. We subjected the oral isolates to 14 different biochemical tests. The biochemical abilities of SJM-04, SJM-38, and SJM-65, to me-

Biochemical Characterizations of the Three Oral Isolates
The survival of oral communities largely relies on the nature of the salivary environment (pH, temperature, and ionic strength) and the intrinsic metabolic responses of these communities to the salivary biochemical milieu. We subjected the oral isolates to 14 different biochemical tests. The biochemical abilities of SJM-04, SJM-38, and SJM-65, to metabolize monosaccharide and disaccharide and produce citrate, cytochrome oxidase, nitrate reductase, amylase, and lipase were determined using Voges-Proskauer (VP), sucrose fermentation, citrate, catalase, nitrate reductase, starch, and lipid hydrolysis assays, respectively ( Table 2). According to Kampfer et al. [33], most strains of Georgenia species are able to utilize glucose and sucrose, and also showed that a Gram positive coccoid Georgenia sp. (~1-1.5 mm) isolate with a positive oxidase reaction demonstrated aerobic metabolism. The isolate SJM-38, identified as S. saprophyticus, a Gram positive cocci, is commonly found in the female urinary tract [34], but has also been isolated from meat, raw milk, cheese products [35], and the marine environment in polluted and recreational waters [32,36]. Recently, Uttatree and Charoenpanicha [37] reported certain biochemical properties of S. saprophyticus including fermentation and oxidation of glucose and sucrose, as we detected in the current study. Additionally, S. saprophyticus strains exhibited the production of citrate, catalase, amylase, and lipase ( Table 2). The negative reaction of VP was well supported by the literature [38]. At least four types of nitrate-reducing enzymes have been reported in oral microflora, (i) periplasmic (NAP), (ii) membrane-bound (NAR), (iii) ferredoxin-dependent assimilatory (FdNAS), and (iv) flavin-dependent assimilatory (FAD-NAS), which exhibit distinct biochemical and catalytic properties of bacterial species including R. mucilaginosa, R. dentocariosa, and S. epidermidis [39]. Moreover, the isolate SJM-65 R. mucilaginosa was found to share a positive catalase reaction and a coccoid morphology with Staphylococci species [40]. Recently, Dhital et al. [41] reported a common starch hydrolytic reaction, whereby oral isolates secrete amylolytic enzymes that convert complex starch oligomers into simpler forms.

Effects of Temperature, pH, and NaCl on Biofilm Formation
Growth patterns of bacterial cells proportionally affect the growths of biofilms, which are largely composed of non-replicating persister cells in an extracellular polysaccharide

Effects of Temperature, pH, and NaCl on Biofilm Formation
Growth patterns of bacterial cells proportionally affect the growths of biofilms, which are largely composed of non-replicating persister cells in an extracellular polysaccharide (EPS) matrix [43,44]. Unlike free planktonic cells, biofilm embedded/phenotypically altered cells become acclimatized to withstand microenvironmental stresses such as temperature, pH, and ionic strength changes. Hence, the present study primarily ascertains the optimal biofilm formation by modulating the physiochemical growth conditions for Georgenia sp. (SJM-04), S. saprophyticus (SJM-38), and R. mucilaginosa (SJM-65). Our results demonstrated ( Figure 3A) that a lower temperature (28 • C) had a negligible effect on biofilm formation by bacterial strains as compared with the control temperature (37 • C). However, temperature elevation to 42 • C significantly limited biofilm adherence to 7.2 ± 1.0% (p < 0.05) for strain Georgenia sp. (SJM-04), and S. saprophyticus (SJM-38) and R. mucilaginosa (SJM-65) could not survive this temperature. It is widely accepted that the optimum temperature is directly related to the metabolic activities of microbial enzymes, and thus, nutrient metabolism [45] and biofilm formation [46].

Physicochemical Characteristics of TiO2NPs
Surface plasmon resonance (SPR) happens due to the collective oscillations of electrons at the resonant frequency of metal NPs and results in absorption in the UV-Visible region [13]. In the present study, the appearance of a sharp peak at an absorption wavelength (λmax) of 347 nm in the UV-Vis absorption spectrum is likely to be due to localized SPR of TiO2NPs in aqueous suspension ( Figure 4A). Furthermore, SPR frequencies of NPs In the present study, an increase or decrease in one pH unit from the control level (pH-7.4) significantly (p < 0.05) affected the interaction between isolates Georgenia sp. (SJM-04), S. saprophyticus (SJM-38), and R. mucilaginosa (SJM-65) and glass surfaces ( Figure 3B). At pH 6.4, significant (p < 0.05) increases in biofilm formation were observed for Georgenia sp. (SJM-04), S. saprophyticus (SJM-38), and R. mucilaginosa (SJM-65) strains by 105.2%, 120.7%, and 166%, respectively, as compared to 100% for controls at pH 7.4). Conversely, an increase in pH to 8.4 caused significant (p < 0.05) reductions in biofilm to 85.77 ± 0.80, 73.26 ± 3.6, and 88.57 ± 4.2%, respectively ( Figure 3B). Thus, our results indicate that a slight change in external pH can overwhelm the cellular processes that support oral bacterial biofilms, which may include the synthesis of proteins [47] and polysaccharides [48] and the membrane electrochemical gradient [49]. Earlier studies on acyl-homoserine lactone (AHL) production in quorum sensing (QS) systems of marine bacteria demonstrated salinity dependence [50], and suggested that salinity is a significant factor for QS [51]. Therefore, we also investigated biofilm growth in the presence of different concentrations (200, 400, 600, 800, and 1000 µg/mL) of NaCl. Results demonstrated a dose-dependent decrease in biofilm formation from 98.5 ± 2.5% to 90.0 ± 8.7% and from 76.9 ± 2.8% to 59.7 ± 1.2% on increasing the NaCl concentration from 200-1000 µg/mL for Georgenia sp. (SJM-04) and R. mucilaginosa (SJM-65), respectively ( Figure 3C). Interestingly, under identical conditions, biofilm formation by S. saprophyticus (SJM-38) slightly increased on increasing the NaCl (200-1000 µg/mL) concentration 101.3 ± 1.2%, 107.7 ± 1.6%, 110.3 ± 0.7%, 104.4 ± 0.7% and 100.4 ± 1.6%, respectively ( Figure 3C). According to Moretro et al. [52], NaCl and glucose stimulate adherence and increase the stability of biofilms formed by Staphylococci genus due to the presence of the icaA gene, which is positively correlated with strong biofilm formation. Recently, Xu et al. [53] also reported that NaCl significantly increased biofilm formation by S. aureus in an concentration-dependent manner

Physicochemical Characteristics of TiO 2 NPs
Surface plasmon resonance (SPR) happens due to the collective oscillations of electrons at the resonant frequency of metal NPs and results in absorption in the UV-Visible region [13]. In the present study, the appearance of a sharp peak at an absorption wavelength (λ max ) of 347 nm in the UV-Vis absorption spectrum is likely to be due to localized SPR of TiO 2 NPs in aqueous suspension ( Figure 4A). Furthermore, SPR frequencies of NPs are considered to be directly correlated with nanoparticle size, shape, and crystalline nature [54]. Therefore, we analyzed the morphology and composition and determined the size and crystallinity of TiO 2 NPs. SEM analysis showed NPs had pleomorphic shapes, though the majority were spherical ( Figure 4B). The EDX spectrum of TiO 2 NPs revealed the presence of titanium (Ti) and oxygen (O) at elemental compositions of 32.74% and 67.26%, respectively ( Figure 4C). TEM results showed TiO 2 NPs shapes included spherical, oval, and hexagonal particles ( Figure 4D) with sizes ranging from 5-25 nm (average diameter 14.7 nm) ( Figure 4E). Furthermore, the XRD pattern of TiO 2 NPs ( Figure 4F), obtained by using cell parameters: a-3.8101 Å, b-3.8101 Å, and c-9.3632 Å; α = β = γ = 90 • and centered tetragonal phase, showed the anatase phase ameter 14.7 nm) ( Figure 4E). Furthermore, the XRD pattern of TiO2NPs ( Figure 4F), obtained by using cell parameters: a-3.8101 Å, b-3.8101 Å, and c-9.3632 Å; α = β = γ = 90° and centered tetragonal phase, showed the anatase phase TiO2-NPs (JCPDS 21-1272) and peaks at 2θ values of 24.6°, 37.2°, 47.5°, 53.4°, 54.6°, and 62.2° corresponding to (101), (004), (200), (1050), (211), and (204) HKL miller indices, respectively. Average size by XRD was determined to be 13.7 nm based on full-width at half-maximum (FWHM) of the 101 reflection peak, which matched well with that of the TEM size.  Figure 5(CI)), respectively. The mechanisms responsible for the antibacterial activities of various metal-oxide NPs are unclear, though it is believed that the presence of dissolved metal ions on surfaces of NPs and/or NP-induced oxidative stress are involved [14]. Specifically, in the case of anatase TiO2NPs, the photocatalytic activity of TiO2 in aqueous environments results in the release  Figure 5(CI)), respectively. The mechanisms responsible for the antibacterial activities of various metal-oxide NPs are unclear, though it is believed that the presence of dissolved metal ions on surfaces of NPs and/or NP-induced oxidative stress are involved [14]. Specifically, in the case of anatase TiO 2 NPs, the photocatalytic activity of TiO 2 in aqueous environments results in the release of hydroxyl radicals (OH•) and the subsequent formation of superoxide radicals (O 2 − ) [55]. Therefore, it could be argued that ROS may attack polyunsaturated phospholipids in bacteria and cause DNA damage [23,25]. Additionally, we treated Georgenia sp. (Figure 5(AIII)), S. saprophyticus ( Figure 5(BIII)), and R. mucilaginosa ( Figure 5(CIII)) with TiO 2 NPs at 250 µg/mL and examined their effects by SEM. We observed that TiO 2 NPbacteria interactions caused morphological changes such as shrinkage and cell membrane damage, possibly because NPs penetrated bacterial membranes and compromised cell membrane permeability [56]. [55]. Therefore, it could be argued that ROS may attack polyunsaturated phospholipids in bacteria and cause DNA damage [23,25]. Additionally, we treated Georgenia sp. ( Figure  5(AIII)), S. saprophyticus ( Figure 5(BIII)), and R. mucilaginosa ( Figure 5(CIII)) with TiO2NPs at 250 µg/mL and examined their effects by SEM. We observed that TiO2NP-bacteria interactions caused morphological changes such as shrinkage and cell membrane damage, possibly because NPs penetrated bacterial membranes and compromised cell membrane permeability [56].

Dose-Dependent Effects of TiO2NPs on Biofilm Formation
The effects of TiO2NPs concentration (250-2000 µg/mL) on the adherence of the biofilms produced by the three oral strains were also examined. The biofilms produced by various bacterial species play decisive roles in the way they respond to their immediate surroundings. Treatment of Georgenia sp., S. saprophyticus, and R. mucilaginosa with TiO2NPs at 250, 500, 1000, or 2000 µg/mL for 24 h significantly reduced biofilm adhesion on glass surfaces to 55 Figure 6A). Taken together, these results show that TiO2NPs reduced biofilm adherence in a concentration-dependent manner. Recently, Sodagar et al. [57] reported that treatment with 5% TiO2NPs significantly inhibited biofilm formation by the Gram positive oral bacteria S. mutans and S. sanguinis. Additionally, the micrographs presented in Figure 6B-D show than TiO2NPs reduced biofilm formation by Georgenia sp. (Figure 6(BII-BV)), S.

Dose-Dependent Effects of TiO 2 NPs on Biofilm Formation
The effects of TiO 2 NPs concentration (250-2000 µg/mL) on the adherence of the biofilms produced by the three oral strains were also examined. The biofilms produced by various bacterial species play decisive roles in the way they respond to their immediate surroundings. Treatment of Georgenia sp., S. saprophyticus, and R. mucilaginosa with TiO 2 NPs at 250, 500, 1000, or 2000 µg/mL for 24 h significantly reduced biofilm adhesion on glass surfaces to 55 Figure 6A). Taken together, these results show that TiO 2 NPs reduced biofilm adherence in a concentration-dependent manner. Recently, Sodagar et al. [57] reported that treatment with 5% TiO 2 NPs significantly inhibited biofilm formation by the Gram positive oral bacteria S. mutans and S. sanguinis. Additionally, the micrographs presented in Figure 6B-D show than TiO 2 NPs reduced biofilm formation by Georgenia sp. (Figure 6(BII-BV)), S. saprophyticus (Figure 6(CII-CV)), and R. Mucilaginosa (Figure 6(DII-DV)) in a concentration-dependent manner versus untreated controls ( Figure 6(BI,DI)). In assessing the reduction in mature (24 h) biofilms of the three tested strains of TiO 2 NPs by CV assay, only 1000 or 2000 µg/mL resulted in significant destruction of mature biofilms, suggesting that TiO 2 NPs are more efficient against developing biofilms at 250-500 µg/mL, but they can also destroy biofilms at higher concentrations of 1000 or 2000 µg/mL (Supplementary Materials, Figure S1). saprophyticus ( Figure 6(CII-CV)), and R. Mucilaginosa (Figure 6(DII-DV)) in a concentration-dependent manner versus untreated controls (Figure 6(BI,DI)). In assessing the reduction in mature (24 h) biofilms of the three tested strains of TiO2NPs by CV assay, only 1000 or 2000 µg/mL resulted in significant destruction of mature biofilms, suggesting that TiO2NPs are more efficient against developing biofilms at 250-500 µg/mL, but they can also destroy biofilms at higher concentrations of 1000 or 2000 µg/mL (Supplementary Materials, Figure S1).

Conclusions
The MDR Gram positive cocci Georgenia sp., R. mucilaginosa, and S. saprophyticus isolated from oral cavity were successfully characterized for their morphologies, biochemical characteristics, phylogenetic relatedness, and biofilm formation at various pH, temperatures and salt concentrations. Exposure of these strains to crystalline TiO2NPs (5> size <25 nm) significantly inhibited their planktonic cell growth and biofilm formation. Three exposure scenarios including low (250 µg/mL), moderate (500 µg/mL), and high (1000-2000 µg/mL) doses of TiO2NPs decreased the biofilm in a dose-dependent manner, suggesting that the concentration of TiO2NPs, apart from other factors, could be the main reason why they act as both an antibacterial and antibiofilm agent to the tested oral bacteria. Our results suggest that TiO2NPs with the following physicochemical profile: absorption λmax of 347 nm, diameter 5-25 nm, average crystalline size 13.7 nm, tetragonal anatase phase, and spherical shape might be a suitable choice for treating oral biofilms, can potentially be applied in orthodontics as a potential oral hygiene alternative to conventional rinses and for the suppression of cariogenic biofilm formation. Further in vivo biofilm studies on the interaction of TiO2NPs with human saliva and the effect on NP's shape, size, and metal release are warranted for preparing the most effective antibiofilm formulations.

Conclusions
The MDR Gram positive cocci Georgenia sp., R. mucilaginosa, and S. saprophyticus isolated from oral cavity were successfully characterized for their morphologies, biochemical characteristics, phylogenetic relatedness, and biofilm formation at various pH, temperatures and salt concentrations. Exposure of these strains to crystalline TiO 2 NPs (5> size <25 nm) significantly inhibited their planktonic cell growth and biofilm formation. Three exposure scenarios including low (250 µg/mL), moderate (500 µg/mL), and high (1000-2000 µg/mL) doses of TiO 2 NPs decreased the biofilm in a dose-dependent manner, suggesting that the concentration of TiO 2 NPs, apart from other factors, could be the main reason why they act as both an antibacterial and antibiofilm agent to the tested oral bacteria. Our results suggest that TiO 2 NPs with the following physicochemical profile: absorption λ max of 347 nm, diameter 5-25 nm, average crystalline size 13.7 nm, tetragonal anatase phase, and spherical shape might be a suitable choice for treating oral biofilms, can potentially be applied in orthodontics as a potential oral hygiene alternative to conventional rinses and for the suppression of cariogenic biofilm formation. Further in vivo biofilm studies on the interaction of TiO 2 NPs with human saliva and the effect on NP's shape, size, and metal release are warranted for preparing the most effective antibiofilm formulations.