Antibacterial Applications of Anatase TiO2 Nanoparticle

Many methods have been used for the preparation of nanostructured metal oxides. Here we report the synthesis of TiO2 nanoparticles by facile hydrothermal process by varying the concentration of the precursor and reaction temperature while keeping the process time constant. Morphological, structural and optical studies were carried out by scanning electron microscopy equipped with energy dispersive spectroscopy, X-ray powder diffraction spectroscopy and VU-Vis-NIR spectroscopy. Morphological and compositional analysis reveal that the prepared nanoparticles are highly pure with an approximate average particle size 5-15nm. XRD studies showed their crystalline structure and the sizes around 5 nm, while the optical absorption studies in the photon wavelength range 300-600 nm reveal that the strong absorbance peak is positioned at around 3.5 eV nm whereas visible energy is almost transparent for the materials. Finally, the antibacterial effect of TiO2 nanoparticles has been studied. Plating technique was used to determine lowest concentration that prevent or inhibit growth of bacteria. This technique of TiO2 nanoparticle was used against most common organisms which cause wound infection including: MRSA, E. coli and Pseudomonas aeruginosa. Different concentrations of TiO2 nanoparticles were used 100 μg/ml, 200 μg/ml, 400 μg/ml, 600 μg/ml and 800 μg/ml. Inhibition of bacteria was different for prepared TiO2 samples due to different concentration of the precursors and synthesis temperature.


Introduction
In the recent past, nanomaterials of different metal oxides have attracted much attention due to their variety of technological application they offer such as, catalysis, sensors, optoelectronics applications, solar cells, electroluminescent devices, light-emitting devices and environmental remediation [1][2][3][4][5][6][7]. Researchers have always been interested to synthesize different nanomaterials and exploit them in diverse applications to improve the quality and efficiency of the devices. As a matter of fact, nanoparticles (NPs) possess altogether distinctive properties compared to their bulk materials. Apart from their application in optoelectronics and environmental, nanoparticles have also been equally improving the quality of human life by exploiting them in life sciences. Metal oxide nanoparticles are of great interest for scientists to use them in medicine, pharmaceutical and biological applications [8,9]. Due to their low dimensions and high surface to volume ratio, NPs attach firmly with microbial membranes which play pivotal role for antibacterial efficiency [10]. In biomedical devices, the NPs are coated in the form of oxides to prevent bacterial proliferation and colonization due to their catalytic activity. They have successfully been exploited for the detection of proteins, production of drugs, purification and separation of cells and biological molecules and for the cure of tumors [11].
TiO 2 NPs have emerged as new generation of novel materials and of prime interest due to its applications in vast area of nanoscience because it is non-toxic and cost effective. The physical properties of TiO2 NPs largely influence their applications [12]. It is found in three forms, i.e., brookit, anatase and rutile. Out of three, anatase NPs are commonly used for different applications such as, dye sensitized solar cells, gas sensors, photo degradation of organic compound, organic synthesis, deactivation of microorganisms and cell culture [13]. The restrictive performance of NPs generally follows mainly two pathways that are colligated to each other and may occur at the same instant. First, interruption of membrane potential and integrity and secondly, production of oxygen free radicals, i.e., NPs are acting as nanocatalyst [14]. TiO 2 NPs have been successfully synthesized by several methods, for example, sol-gel, precipitation, electrochemical, solvothermal, sonochemical, solid state reactions and microwave irradiation technique [15][16][17][18][19][20][21]. These processes involve environmentally unfriendly reagents which may cause biological hazards or toxicity.
In this study, TiO 2 spherical nanocrystallites with anatase crystal structures are synthesized by facile hydrothermal process, chemically and physically characterized, and their antibacterial activity has been investigated. The nanoparticles are tested in a dose response strategy against three of the most common pathogenic bacteria: Methicillin Resistance Staphylococcus aureus (MRSA), E. coli and Pseudomonas aeruginosa that cause wound infection. The impact of this work will give new strategies in treatment of wound infection.

Preparation of TiO 2 Nanoparticles
Hydrothermal method has been known as an effective strategy for preparation of oxide nanoparticles both of binary and ternary compounds. This method has some advantages such as, low coast, low temperature, and simplicity of the setup. On the other hand, hydrothermal method has an important disadvantage which is the longtime of heating in autoclave. One of the methods of preparation can overcome of this disadvantage which is heating by microwave radiation. Preparation of nanostructures by microwave heating has more than one of advantages such as, increase of crystallinity without any additional post heat treatments, heating the materials under reaction need short time, consequently produce nanostructures with time and cost saving.
TiO 2 nanostructures were prepared by microwave heating systems. Microwaves have lower energy relative to other forms of electromagnetic energy and lie in frequency range from 300MHz to 300GHz corresponding to energies from 1.24×10-3 meV to 1.24 meV. Heating materials with microwave radiation take place by volumetric absorption of the lower electromagnetic energy and transform into heat of the material under expose. By this way, the generated heat inside the material not like conventional heating where the heat is distributing through the material from outside to inside by conduction. Consequently, the method of heating the materials by microwave has some advantages like as reducing processing time due to fast heating rate and homogeneity in heating process which lead to enhancing quality of material and compound produce by this method. TiO 2 nanoparticles were prepared by hydrothermal condition as follow. Started with the solution of 0.08 M of Ti[OCH(CH 3 ) 2 ] 4 in de ionized water. The final solutions were adjusted to 25 mL in 50 mL Teflon vessel by adding Di-water, followed by sealing the vessel. The reactions for different samples were conducted at different temperatures in microwave oven and time was kept constant as 90 min, then subsequently, cooled to the room temperature slowly. Finally, TiO 2 nanopowders were collected by centrifugation and washed by water five times. The final powders dried in desiccator. Different conditions were used for preparation of TiO 2 and listed in Table 1.

Growth and Morphological Characteristic of Bacterial Strains
The growth of Staphylococcus aureus subsp. aureus (ATCC® 43300MINIPACK™), Pseudomonas aeruginosa (ATCC® 27853™) and Escherichia coli (ATCC® 25922™) were obtained from American Type Culture Collection (ATCC) org. (Manassas, USA). The bacterial strains were characterized by observing the optical density (OD) and colony forming units (CFUs) of the bacterial cells over time. Aseptic methods are necessary for managing all the microbiological works, which was achieved under laminar flow hood. A stock cultures of the bacteria were stored at (-20°C) in Trypticase Soy Agar/Broth (TSB) supplemented with 50 % sterile glycerol which used as cryopreservative. The bacterial strains were sub-cultured onto Luria Bertani (LB) agar plate from a 200 μL frozen glycerol stock by using a sterile inoculation loop. The sub-culture was incubated overnight at 37o C for 18 hours minimum. The LB ager and broth were purchased from Micromaster Laboratories Pvt. Ltd. (Maharashtra, India). The LB agar used for growing strains was supplemented with 10 gm/L casein enzymes hydrolysate, 5 gm/L yeast extract, 5 gm/L NaCl and 15 gm/L agar. Similar ingredients are existing in LB broth without agar. A 40 g of LB agar powder was dissolved in 1000 ml distilled water while preparation of LB broth was required 1 g of powder dissolved in 50 ml distilled water. The media of LB agar and broth were sterilized at 121°C in autoclave, then the agar medium was poured into sterilized petri-dishes and kept at room temperature for solidification. From the first agar plate, a single colony was streaked out on a new LB agar plate for preparing a second sub-culture which was incubated at 37°C overnight. For bacterial inoculum, a single colony from second sub-culture plate was inoculated in 100 ml Erlenmeyer flask containing 50 ml LB broth. The inoculum was incubated in a rotary shaker (GFL 3031 Shaking Incubator from UNIQUE Medical Laboratory Equipment Trading & Services, Sharjah, United Arab Emirates) at 37°C at speed 150 rpm for 18 hours. The optimum growth of the overnight culture was observed by using a spectrophotometer (GENWSYS™10S UV-VIS Spectrophotometer from Thermo Fisher Scientific Inc., Madison, USA) to measure the optical density at 600 nm (OD600).

Preparation of Bacterial Suspension
The bacterial growth was monitored by measuring the turbidity of the LB broth media at OD600. After the overnight incubation, the bacterial cells were transferred into 50 ml polypropylene conical VWR® high-performance centrifuge tubes with flat or plug caps (VWR International, LLC Radnor, PA, USA) and harvested by centrifugation (High-Speed Refrigerated Centrifuges CR 22 GIII from Hitachi Koki Co., Ltd., Japan) at 5,000 rpm for 7 minutes at room temperature (22°C). The supernatant was discarded and the harvested pellet was washed three times with 10 ml of 0.9 % NaCl normal saline, which was purchased from Pharmaceutical Solutions Industry (PSI) (Jeddah, Saudi Arabia). The pellet in each washing were centrifuged for 7 minutes at 5,000 rpm at room temperature (22°C. After the third washing, the microbial stock solutions were prepared by re-suspending the final pellets in 10 ml of 0.9 % NaCl normal saline. Absorbance of the suspension was measured at OD600 to count the viable cells before adding the TiO 2 nanostructures [22].

Colony Counting of Bacterial Cells
The bacterial cells were divided into six tubes; each contains 5 ml of bacterial suspension. TiO 2 nanocrystallites were weighed at dose response manner (100, 200, 400, 600 and 800) μg using an analytical laboratory balance (XPE504DR from Mettler-Toledo International Inc, USA). There were several methods for nanoparticles sterilization. Thus, after measuring the different TiO 2 nanocrystallites doses, they were sterilized using UV light for 45 minutes [23]. The Control suspension was used without TiO 2 nanocrystallites. TiO 2 nanocrystallites were dissolved in 5 ml bacterial suspension at dose response manner and mix gently by vortex (Bench Mixer Benchmark Vortex Mixer from Science Lab Supplies, St. Augustine, FL). The bacterial growth was examined by measuring the OD600 after adding the TiO 2 nanocrystallites. Fritz et al. described the drop-plating method for counting the CFUs of bacterial cells [24]. At zero time, serial dilutions (1:10) from (10-1-10-8) were accomplished by diluting the bacterial cells (100μL) in micro-centrifuge tubes (SPL Life Sciences,Pocheon-si, Gyeonggi-do, Korea) consisting of sterile 0.9% NaCl normal saline (900μL) [25]. For the plating method, three 10μL aliquots of the proper dilution were dotted onto LB agar using an automatic pipette and incubated overnight at 37°C. The samples were reincubated in a rotary shaker at 37°C at speed 150 rpm for several time intervals.

Growth Curves of Bacterial Cells Exposed to TiO 2 Quantum Dots
Bacterial cells growth happens in five phases: lag phase, exponential phase, stationary phase, death phase and extended or long-term stationary phase [26]. The growth curve was measured both viable and dead bacteria. The lag phase is the most unwell described phase and has been supposed to be a process of absence of bacterial growth for a period that's required to achieve the new medium. Some aspects may affect the bacterial growth during lag phase that vary among bacterial species or the period of cells stayed at starved conditions [27]. On the other hand, the exponential phase and stationary phase were representing the rate of cell division and termination of cell division, respectively. Stochastic death and programmed cell death was resulted when bacterial cells exhausted the nutrients in the medium. At this phase, the nutrients were released into environment from dead cells which allowed the survivors bacteria to use these constituents for their survival and entering in long-term stationary phase [28]. The bacterial cells from long-term stationary phase are capable of maintaining their constant cell density and stay viable for years in a batch culture without supplements [29]. The cells inoculated from this phase were revealed an extended lag phase in growth compared to their growth from late stationary phase [28]. Hence, the TiO 2 quantum dots were added at different doses to measure their effect on the growth of bacterial cells. At 1-hour time intervals, serial dilution was performed as previously described at time zero for samples recovered from the shaker and the growth was checked by measuring the OD600. Drop-plate method was implemented for the recovered samples by spotting 10μL aliquots in triplicates on LB agar and incubated at 37°C overnight. Samples were re-incubated in a rotary shaker at 37°C at speed 150 rpm which the similar protocol of dilution, drop-plating and measuring the OD600 was done for 2 hours, 3 hours and 24 hours. The experimental procedures were achieved for negative-control (without TiO 2 nanocrystallites) and the treatment samples (containing TiO 2 nanocrystallites) [30].

Determination of Minimum Exposure Time of Efficient Antimicrobial Activity of TiO 2 nanocrystallites
The dose response curve experiment was completed after the 24 hours when bacterial cells reached to the decline phase. The antimicrobial activity of bacterial strains against a range of known concentration of TiO 2 nanocrystallites after a period of time intervals was evaluated by counting the CFUs/ml to detect to minimum inhibitory concentration (MIC). The MIC was taken at a specific concentration required to arrest bacterial growth at a specific time. The bacterial growth was determined by plotting dose response curve of CFUs/ml and OD versus time. A comparison was made from the dose response curves to investigate the ideal TiO 2 nanocrystallites concentration that showed the antimicrobial effect at a certain exposure time [30].

Characterization
The morphology of prepared powder samples was analyzed by field emission scanning electron microscopy Jeol, Japan (FESEM-JSM-7600F), chemical compositions were measured by energy dispersive spectroscopy using a FESEM attached with an Oxford EDS system. Whereas, the X-ray diffrectometer was used to analyze the crystalline nature and phase studies of the undoped and Zn-doped samples. Rigaku, Ultima-IV was used to perform the XRD measurements which is equipped with Cu-Kα radiation (λ=1.54060 nm) operated at 40 kV and 40 mA at room temperature. The XRD spectra were measured in the 2θ angular region between 10° to 80° and step size was kept 0.05°. Optical absorption spectra were measured using a UV-Vis-NIR spectroscopy (Jasco V-770 attached with 60 mm integrating sphere) in the spectral range 200-800 nm. Finally, plating technique was used to analyse the antibacterial activity of the prepared TiO 2 nanoparticles samples.

Surface Morphological Studies
Surface morphological studies of the prepared TiO 2 samples (Ti1-4) were performed by scanning electron microscopy. Figure 1 shows the typical high magnification images of the as prepared TiO 2 nano-powder samples deposited on carbon stripe. Around the examining area, it can be noticed that the nanoparticles are densely aggregated in the form of big clusters but the approximate average size of the nanoparticles is 5-15nm and are semi spherical in shapes. It can be easily noticed from the SEM micrographs that except samples Ti1 where the particles size is little smaller than rest of the samples (Ti3, and Ti4) which were prepared at low temperature and high concentration and size of Ti1 sample also smaller than the sample high temperature and low concentration. However, over all there is small variation in the particle sizes of all four samples which may be due to the conditions applied for their synthesis.   Table 2. Clearly, the samples that prepared at low temperature and that prepared with low concentrations have smaller O/Ti weight percentage ratios relative to the samples prepared at high temperatures with high concentration of Ti ions in the solution (Ti4).

Structural Analysis
The XRD patterns of different samples of TiO 2 obtained are shown in Figure 3. All the spectra clearly show the polycrystalline structure of the TiO 2 nanoparticles with different orientation along different planes. The diffraction lines in the XRD spectra are matching to the anatase crystalline phase of TiO 2 which are well indexed with the reference card No. ICCD-PDF 44309. It can be noticed in the spectra that only anatase phase is present in the sample. The broad peaks in the spectra indicate that the particle size is relatively small.

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The peak intensities of the samples Ti3 is higher than those of Ti1, 2, 4, samples which may be due to slight changes in the grain size of the TiO 2 . Crystallite size of the TiO 2 nanoparticles could be estimated using the following Scherrer equation: where D is the crystallite size, K is the Scherrer constant equals to 0.9 for spherical shapes, λ is wavelength of the X-rays radiation, β is the full width at half maximum (FWHM) and θ is the Bragg's diffraction angel.

Optical Studies of the TiO 2 Nanoparticles
Optical absorbance is a powerful method to determine the energy gap and particle size as well as optical properties of the samples. The useful semiconductors for photocatalysis have a band gap comparable to the energy of the photons of visible or ultraviolet light, having a value of Eg < 3.5 eV. The majority of authors have determined that in TiO 2 the rutile has a direct band gap of 3.06 eV and an indirect one of 3.10 eV and the anatase has only an indirect band gap of 3.23 eV [31,32]. However, Reddy "s work shows that a bandgap of anatase phase from the plot for indirect transition are quite low (2.95 -2.98 eV) [31]. Contrary to the other authors, to conclude that the direct transition is more favorable for TiO 2 nanoparticles with anatase phase. There have been reported values in the literature from 2.86 to 3.34 eV for the anatase phase. Here in, UV-Vis absorption spectra were taken in the photon wavelength range between 300 and 600 nm. Figure 4 shows the UV-Vis absorption spectrum of hyderothermally synthesized TiO 2 nanoparticles. As in Figure 4 the optical absorption peak has been found at between 350 nm to 362 nm. The band gaps were obtained from the second derivative of the absorption spectra, and are listed in Table 2.
Clearly, the band gaps increasing with decreasing nanoparticles size and Ti1 sample with smallest nanocrystallite size has the largest band gap. The band gap of the nanocrystallites are blue shifted relative to bulk band gap of TiO 2 . This result in agreement with the previous results that showed the existence of the blue shift for TiO 2 nanocrystallites [33].

Plating Technique for Bacteria Cell
Plating technique was used to determine lowest concentration that prevent or inhibit growth of bacteria. This technique of TiO 2 nanoparticle was used against most common organisms that's cause wound infection including: MRSA, E. coli and Pseudomonas aeruginosa. Different concentrations of TiO 2 were used 100 μg/ml, 200 μg/ml, 400 μg/ml, 600 μg/ml and 800 μg/ml. Inhibition of bacteria was different between the samples under investigation due to different physical properties and Ti/O ratios.

Antibacterial Activity of TiO 2 Nanoparticles
In this work, we used TiO 2 nanoparticles which were synthesized with novel method, and produced four samples with different O/Ti ratio. Each concentration was assayed individually in order to assess inhibitory activity against MRSA, E. coli and Pseudomonas aeruginosa and dose response manner was created.

Sample Ti1 of TiO 2 Nanoparticles
Antibacterial activity of TiO 2 nanoparticles were investigated against wound pathogens MRSA, E. coli and Pseudomonas aeruginosa. Experiments were performed with different concentrations at various times to determine which one of strain is effective at ambient conditions. Fig.  5 displays gradual increase of inhibition of MRSA up to 120 minutes for 100 μg/ml and 200 μg/ml concentration then inhibition was sharply increased at time 180 minutes. After that, slight increase was noticed at time 1440 minutes. However, inhibition with concentrations 400 μg/ml, 600 μg/ml and 800 μg/ml were increased gradually until 1440 minutes. Inhibitions of Pseudomonas aeruginosa with Ti1 sample revealed favorable effect against the growth of bacteria. At concentrations 100 μg/ml, 200 μg/ml and 400 μg/ml inhibition were increased sharply up to 120 minutes and then gradually increased up to 1440 minutes. Whereas at time 60 minutes, concentrations 600 μg/ml and 800 μg/ml exhibited highest inhibition of bacteria, then it was slightly increased up to 1440 minutes. E. coli exhibit also gradual inhibition until it reaches to 180 minutes then inhibition was increased sharply after 180 minutes for concentration 100 μg/ml until time 1440 minutes. Regarding the other concentrations 200 μg/ml, 400 μg/ml, 600 μg/ml and 800 μg/ml, good inhibition was observed at time 60 minutes then slightly decrease in inhibition was observed until time 180 minutes. After that, the inhibition was increased until time 1440 minutes Figure 5.

Sample Ti2 of TiO 2 Nanoparticles
Sample Ti2 of TiO 2 nanoparticles showed different effects with pathogenic strains that cause wound infection as shown in Figure 6. With this sample Ti2, MRSA exhibited few inhibitions at time 0 minutes, then the effect of Ti2 of TiO 2 nanoparticles was slightly decreased until time 120 minutes for concentrations 100 μg/ml, 200 μg/ml, 400 μg/ml, 600 μg/ml and 800 μg/ml. Then the inhibitory effect was increased again gradually until time 1440minutes. Regarding the inhibition of Pseudomonas aeruginosa by Ti2 sample concentrations 100 μg/ml, 200 μg/ml, 400 μg/ml, 600 μg/ml showed maximum inhibition until time 180 minutes. Then inhibitory effect was slightly decreased at time 1440 minutes. However, concentration 800 μg/ml showed highest inhibition at time 120minutes, then the inhibitory effect of TiO 2 shows minor increase until time 1440 minutes. E. coli displays different effect with each concentration, as with concentration 100μg/ml sharp inhibition of bacterial growth was observed until time 120 minutes. After that inhibitory effect was slightly decreased at time 180 minutes, then inhibition was increased again at time 1440 minute for concentration 100 μg/ml. No any effect was noticed for concentration 200μg/ml at time 0 minutes, however, after 60 minutes inhibitory effect was sharply increased until 120 minutes followed by slight increase until 1440. Other concentrations 400 μg/ml, 600 μg/ml and 800 μg/ml showed best inhibitory effect until time 60 minutes, then the inhibition was increased slightly until time 1440 minutes which has been showed Figure 6.

Sample Ti3 of TiO 2 nanoparticles
Regarding the sample Ti3 of TiO 2 nanoparticles, sharp inhibitory effect was observed on MRSA until time 60 minutes with all concentrations 100 μg/ml, 200 μg/ml, 400 μg/ml, 600 μg/ml, and 800 μg/ml. Then inhibition was gradually increased until time 1440 minutes showed in Figure 7. With Pseudomonas aeruginosa maximum inhibitory effect was observed for all different concentrations 100 μg/ml, 200 μg/ml, 400 μg/ml, 600 μg/ml and 800 μg/ml until time 120 minutes. After that, with concentrations 100 μg/ml and 800 μg/ml showed gradual increased in inhibitory effect until time 180 minutes followed by slight decrease at time 1440 minute. Regarding the concentrations 200 μg/ml, 400 μg/ml and 600 μg/ml showed little decreased in inhibitory effect until time 1440 minutes. In E. coli, inhibitory effect was gradually increased until time 1440 minutes with concentrations 100 μg/ml, 200 μg/ml. Whereas with concentration 400 μg/ml inhibitory effect was gradually increased until time 60 minute, after that it showed minor decreased in inhibition until time120 minutes. A sharp increase was observed until time 180 minutes and then slight increase in inhibition was noticed at time 1440 minutes. Regarding the concentration 600 μg/ml inhibition was slightly increased until time 60 minutes. After that, inhibitory effect was slightly decreased at time 120 minutes. After 120 minutes, inhibitory effect was gradually increased until time 1440 minutes. However, with concentration 800 μg/ml inhibitory effect was increased very slightly until time 180 minutes then inhibitory effect remained the same without any obvious effect until time 1440 minutes.

Sample Ti4 of TiO 2 Nanoparticles
MRSA in sample Ti4 of TiO 2 nanoparticles exhibited gradual inhibitory effect until time 60 minutes with concentrations 100 μg/ml, 200 μg/ml and 400 μg/ml then inhibitory effect of TiO2 nanoparticles was slightly deceased at time 120 minutes. After that, inhibition was gradually increased until time 1440 minutes showed Figure 8.
Gradual inhibitory effect was observed with cconcentration 600 μg/ml until time 1440 minutes. Whereas with concentration 800 μg/ml inhibition was slightly increased until time 120 minutes, then sharply increased at time 180 minutes and finally slight increase was noticed at time 1440 minutes. With Pseudomonas aeruginosa maximum inhibitory effect was observed with all different concentrations 100 μg/ml, 200 μg/ml 400 μg/ml, 600 μg/ml and 800 μg/ml at time 60 minutes. Then the inhibitory effect was increased slightly until time 180 minute. After that, concentrations 100 μg/ml, 200 μg/ml, 600 μg/ml and 800 μg/ml showed little decreased in inhibition at time 1440 minutes, while 400 μg/ml remained without obvious effect Fig. 9. In E. coli highest inhibitory effect was observed until time 60 minutes with concentrations 100 μg/ml, 200 μg/ml, 400 μg/ml, 600 μg/ml and 800 μg/ml. The concentrations 100 μg/ml, 200 μg/ml, 400 μg/ml and 600 μg/ml showed gradual increase in inhibition until time 1440 minutes, whereas with concentration 800 μg/ml inhibitory effect showed minor increased until time 1440 minutes showed Figure 8.

Dose Response Curve
Antibacterial activity of TiO 2 NPs was evaluated against MRSA, E. coli and Pseudomonas aeruginosa by dose response curve analysis. Optical density was measured and plotted as a function of time for 1440 minutes at regular intervals with various concentrations of TiO 2 NPs 100 μg /ml, 200 μg /ml, 400 μg /ml, 600 μg /ml and 800 μg /ml.

Dose Response Curve of Sample Ti1
Dose Response Curve of bacteria that was treated with TiO 2 nanoparticles indicated that, inhibitory effect of TiO 2 nanoparticles was different between species and from sample to sample. Dose response curve of MRSA, E. coli and Pseudomonas aeruginosa treated with TiO 2 sample Ti1 are shown in Figure 9. Dose response curve of MRSA observed that, optical density of bacterial growth was decreased with increasing the concentrations of TiO 2 NPs. In the absence of TiO 2 NPs, optical density was increased, indicating that bacterial growth was increased, but as the TiO 2 NPs concentration was increased, optical density decreased, showing the reduction of bacterial growth rate. With this sample, lowest concentration 100 μg /ml, 200 μg /ml and 400 μg /ml were exhibited high reduction in bacterial growth rate. Dose response curve of Pseudomonas aeruginosa showed that, optical density of bacterial growth was decreased with increase in the concentration of TiO 2 . Resulting the increased reduction of bacterial growth rate. Dose response curve of E. coli showed very little effect of different concentrations of sample Ti1 of TiO 2 nanoparticles on the growth of bacterial cell rate and consequently, optical density of bacterial growth shows very little decreased with increased concentration.

Dose Response Curve of Sample Ti2
Regarding the effect of sample Ti2 of TiO 2 nanoparticles on MRSA, Dose response curve showed that the optical density of bacterial growth was decreased with increasing the concentration of TiO 2 , resulting the increased reduction of bacterial growth rate. The sample concentrations 400 μg/ml and 600 μg/ml exhibited highest reduction of bacterial growth rate shown in Figure 10. Dose response curve of Pseudomonas aeruginosa showed that, optical density of bacterial growth was decreased with increasing the concentration of TiO 2 , resulting in the increased reduction of bacterial growth rate. In case of E. coli, dose response curve showed that the optical density of bacterial growth was decreased with increasing the concentration of TiO 2 , resulting in the increased reduction of bacterial growth rate. Dose Response Curve of MRSA observed that, optical density of bacterial growth was decreased with increasing the concentration of TiO 2 resulting in the increased reduction of bacterial growth rate. Also, Dose Response Curve of Pseudomonas aeruginosa showed the optical density of bacterial growth was decreased with increasing the TiO 2 concentration, resulting in the increased reduction of bacterial growth rate shown Figure 11. Dose Response Curve of E. coli showed that the optical density of bacterial growth was decreased with increasing the concentration of TiO 2 , resulting in the increased reduction of bacterial growth rate. Specially, with concentrations 100 μg/ml, 200 μg/ml and 400 μg/ml, optical density was considerably decreased resulting in the increased reduction growth rate of bacterial cell. In sample Ti4, dose response curve of MRSA showed little effect of different concentrations of sample Ti4 of TiO 2 on growth rate of bacterial cell, resulting in the minor decreasing effect of optical density of bacterial growth with increased concentration. However, dose response curve of Pseudomonas aeruginosa showed, optical density of bacterial growth was decreased with increasing the concentration of TiO 2 , resulting in the increased reduction of bacterial growth rate. Dose Response Curve of E. coli showed very negligible effect of different concentrations on the growth rate of bacterial cell. While, with concentration 200 μg/ml, optical density was considerably decreased compared to the other concentrations, resulting in the increased reduction of growth rate of bacterial cell shown in Figure 12

Discussion
In this work, effect of anatase structure of TiO 2 nanocrystallites with varied sizes on the inhibition of MRSA, E. coli and Pseudomonas aeruginosa at ambient condition were investigated. These kinds of bacteria are the most resistant to most of the antibiotics and can be considered as most effective agents of nosocomial infections. Consequently, introducing the new antibacterial agents can control the rate of morbidity and mortality that caused by the infectious diseases. It's known that bulk TiO 2 is one of the effective materials in killing microbial cells. Also, effect of bulk TiO 2 in killing of cancer cells has also been reported. On the other hand, TiO 2 nanostructure showed a strong inhibiting effect towards a various type of bacterial strains. The nanostructures have distinct properties like increase surface per volume ratio and carrier life time which are effective on inhibition of bacterial activity.
XRD and UV-vis absorption proved a change in particles sizes and optical band gap. The effect of nanocrystallite on inhibition of pathogenic strains that lead to wound infection can be concluded as follow. The nanoparticles with larger sizes are more influencing on inhibition of MRSA bacteria more than the smaller one. However, the smaller nanocrystallites sizes are more effective for inhibition of Pseudomonas aeruginosa and E. coli. As the size of nanocrystallite increase the surface per volume ratio decrease and a gradual change on optical properties and physical properties, consequently the effect of inhibition should change with changing nanoparticles sizes. Also, the increase of activity of inhibition for smaller particles for Pseudomonas aeruginosa and E. coli relative to MRSA inhibition may reflect that the binding of nanoparticless to Pseudomonas aeruginosa and E. coli are different in comparison with the binding of nanoparticless with MRSA. The binding of nanocrystallites with bacterial cells effects on the movement, growth and cell death due to hemostatic imbalance and cellular metabolic disturbance. Moreover, the attachment of nanocrystallite with the cell influence on the production of intracellular reactive oxygen species (ROS). Previous study showed that the coordination and surface properties allow anatase after dispersion induce the generation of ROS [34].
Another important result from the effect of nanoparticless on various kinds of bacteria is the ability of penetration in the cells, we expect that the penetration of smaller nanoparticless into Pseudomonas aeruginosa and E. coli are more effective in comparison with the penetration of smaller nanoparticless into MRSA. The penetration of nanocrysttalite can deactivate biomolecules such as proteins.
According to elemental analysis the nanocrystallites with low O/Ti weight percentage are more effective for inhibition of E. coli and Pseudomonas aeruginosa, which indicates that the increase of Ti ions at the surface of nanocrystallites is one of the factor of antibacterial activity of TiO 2 against Pseudomonas aeruginosa and E. coli strains. However, the nanocrystallites with high O/Ti weight percentage are more effective against inhibition of growth of MRSA, which indicate that the presence of oxygen species on the surface of nanocrystalites is one of the factor that important for antibacterial activity of TiO 2 against MRSA strain.
The effect of TiO 2 concentration on the inhibtion of growth of the three strains MRSA, E. coli and Pseudomonas aeruginosa were investigated. The smallest nanocrystallite size showed a pronounced inhibitory effect and high reduction of growth rate of bacteria with increasing the concentrations of TiO 2 nanocrystallites for the three strains of bacteria.
According to above results the different concentrations of TiO 2 smallest crystallits could inhibit the growth of antibiotic resistant three strains of MRSA, Pseudomonas aeruginosa and E. coli. Consequently, the prepared nanostructures could be considered as excellent alternative treatment for Drug-resistant bacterial infections.