Antimicrobial Potential of Titanium Dioxide Nanoparticles in Urinary Tract Infections: An Experimental Study on the Growth Inhibitory Activity and Biofilm Inhibition

Background: T Microorganisms cause many diseases for the human body such as urinary tract infection and, therefore, it is highly important to eliminate and control them. Bacterial resistance to different types of antibiotics was increased and it is necessary to find alternative agents to eliminate these microbes. Methods: This study aimed was to evaluate the antimicrobial effect of different concentrations of titanium dioxide nanoparticles (TiO2 NPs) on some gram-positive bactria, gram-negative bacteria, and Candida albicans. TiO2 NPs were synthesized using the chemical methods, coated with carboxymethyl cellulose (CMC) and prepared in different concentrations (0.098, 0.196, 0.392, 0.784, 1.568, and 3.136 mg/mL). Eventually, a minimum inhibitory concentration (MIC) and a minimum biofilm inhibitory concentration (MBIC) were applied to investigate the effect of TiO2 NPs on microorganisms. Results: According to the study results, the MICs of TiO2 NPs were found to be 1.489, 1.208, and 1.166 mg/ mL for Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae as the Gram-negative bacteria, respectively; and they were discovered to be 0.512, 0.830, and 0.707 mg/mL for Streptococcus pneumoniae, Staphylococcus aureus, and Staphylococcus epidermidis as the Gram-positive bacteria, respectively. As for C. albicans, as the yeast strain, MIC was 0.253 mg/ mL. The MBIC of more than 90% of TiO2 NPs was 6.25 mg/mL for both Gram-negative and Gram-positive bacterial types and 1.562 mg/mL for C. albicans. Conclusions: It was concluded that TiO2 NPs were effective antimicrobial agents for Gram-positive bacteria, Gram-negative bacteria, and C. albicans, but their inhibitory effect on yeast was greater than that of bacteria.

their high chemical resistance, non-toxicity, long service life, availability, and low cost (11). TiO 2 is widely used in many industries including water purification as well as paints, plastics, paper, and inks manufacturing industries (12). TiO 2 NPs have been applied extensively for killing different groups of microorganisms including bacteria, fungi, and viruses (13). This study, therefore, aimed to synthesize TiO 2 NPs and measure the antibacterial and antifungal effect of different concentrations of them on gram-positive bacteria, gram-negative bacteria, and C. albicans isolated from infected urine samples.

Bacterial Strains
To conduct the present experimental study, three strains of Gram-positive bacteria including Streptococcus pneumoniae PTCC 1240, Staphylococcus aureus PTCC 1431, and Staphylococcus epidermidis PTCC 1114, three Gram-negative bacteria including Escherichia coli PTCC 1399, Pseudomonas aeruginosa PTCC 1690, and Klebsiella pneumoniae PTCC 1290, as well as Candida albicans ATCC 10231 were all purchased from the Iranian Research Organization for Science and Technology.

Sample Collection
As for the sample collection, 60 urine samples (46 male and 14 female cases) were collected from the subjects with typical symptoms of a urinary tract infection. The subjects whose ages ranged from 1.5 to 72 years had been referred to Mohamad Kermanshahi Hospital in Kermanshah, Iran. It should be noted that this study lasted for six months. The men and women referring for the test were advised to clean the genital area before collecting the urine sample, discard the first part of the urine, and ccollect a sample of urine (mid-stream) in the container without stopping the process of urination. Patients' complete information was written on the test tubes. The samples were stored in special containers and their lids were closed and transferred to the university laboratory as soon as possible. Midstream urine samples were quickly cultured on the agar media.

The Identification of Microorganisms
For detecting microorganisms in urine samples, a loop full of samples was shed into EMB, blood agar, and potato dextrose agar and incubated at 37°C overnight. After 24 hours, individual colonies were separated and identified based on the gram-staining, morphological characteristics, and biochemical characters. To detect the gram-negative bacteria, standard biochemical tests were used, including methyl red, citrate, indole, Voges-Proskauer, H 2 S production, oxidase, urea hydrolysis, and gas production, and to detect the Gram-positive bacteria, catalase, coagulase, hemolysis, motility, oxidase, DNase, and mannitol tests. Yeast isolates were identified by the germ tube test and cultured on CHROM agar media. Three strains of gram-positive bacteria including S. pneumoniae, S. aureus, and S. epidermidis, as well as three gram-negative bacteria including E. coli, P. aeruginosa, K. pneumoniae, and Candida albicans were isolated (Tables 1, 2, and 3) in this experimental study (14,15). A microbial solution was obtained from each strain according to the 0.5 McFarland standard (1×10 8 colony forming unit/milliliter for the bacterial strain and 3×10 6 CFU/mL for yeast) to perform the antimicrobial assays (16).

TiO 2 NP Preparation and Coating
In order to carry out this experiment, 5 mL of titanium isopropoxide was added to 8 mL deionized water, and then the precipitation of hydrous TiO 2 and alkoxide was hydrolyzed as well. The resulting solution was kept completely and mixed by persistent stirring at 40°C for half an hour. Finally, the white sediment of TiO 2 NPs was created at the bottom of the tube and then it was separated by centrifuge, washed multiple times with methanol and deionized water, and dried at 80°C in an oven for 12 hours (17,18). To prepare the photocatalytic NPs, the synthesized TiO 2 NPs were first poured into a sterilized  dish and an ultraviolet lamp with a wavelength of 350 nm was placed inside a flask. Then, the output of ultraviolet rays was measured using a digital device. The measured value was 56 µW/m². The UV lamp was then immersed in the NP solution for 60 minutes. Carboxymethyl cellulose (CMC) was used in this research to coat and improve the presentation of the TiO 2 NPs to the target microorganisms as well as to protect the nano agent from degradation (19). TiO 2 NPs were dispersed in 2% CMC and sonicated for 3 hours. For preparing 2% of CMC, 2 g of CMC was mixed with deionized water through mechanical stirring for 12 hours until a perfect dissolution was performed. The morphological and structural characteristics of these TiO2 were analyzed using X-ray diffraction, X-ray microanalysis, transmission, and scanning electron microscopes. TiO 2 NPs used in this study were prepared at a concentration of 25 mg/mL. NPs were diluted (in DMSO-5%) in different concentrations to detect the lethal dose (LD-50). The LD-50 of TiO 2 NP responsible for the 50% death of the tested mice (n= 4 per group) was evaluated by intraperitoneal injection with three concentrations of 0.15 μg/kg (5 mg/mL), 0.3 μg/kg (10 mg/mL), and 0.45 μg/kg (15 mg/mL). Mice were monitored for body temperature, weight, mortality rate, diarrhea, drowsiness, and seizures for 2 weeks. The results of this experiment demonstrated that TiO 2 NPs at these concentrations stimulated no lethal activity after 2 weeks.

Disk Diffusion Method
For determining the antimicrobial effect of nanoparticles, the disk diffusion method was used. Briefly, the Muller-Hinton agar (MHA) medium was prepared and then the plates were inoculated with the sterile cotton swab of microorganisms. The stocks of various concentrations of TiO 2 NP were prepared using dimethyl sulfoxide 2% (DMSO, there was no antimicrobial effect in this dilution), and then 6 mm diameter sterile paper disk, Whatman sterilized filter paper, was used to make the disks, which were impregnated with different concentration of NPs, dried under the laminar hood and sterile condition, and placed on MHA plates. The plates were incubated at 37 º C for 24 hours. The diameter of the inhibition zone, measured as a clear area around the disk, was calculated by a ruler (19). Antibiotic standards adopted in this study were purchased from Padtan Teb Company.

Minimum Inhibitory Concentrations Determination
The antimicrobial effects of TiO 2 NPs were investigated by using the micro-dilution method. In this method, 100 µm of Muller-Hinton broth medium was added to all wells. The NPs were prepared in seven concentrations (0.098, 0.196, 0.392, 0.784, 1.568, and 3.136 mg/mL). The first well in each row was filled with 100 µL of the highest concentration of TiO 2 NPs, and 100 µL of this NP was transferred to the second well, and this process was continued to the 8th well. The 8th well was adopted to control the growth of microorganisms. Finally, microplates were inoculated for 24 hours at 37°C. After that optical density (OD) of these plates was read at 600 nm and inhibitory concentration was reported in the presence of NPs (20).

Minimum Biofilm Inhibitory Concentration (MBIC) Determination
For the determination of anti-biofilm effects of nanoparticles, trypticase soy broth (TSB) medium was supplemented with 1% glucose. Different concentrations of TiO2 NPs were added into the well of 96-well microplates (0.784, 1.568, 3.136, 6.272, and 12.544 mg/ mL). The 24-hours microbial suspension was inoculated in the wells. The microplate was inoculated at 37° C for 24 hours (21). The cultures were then aspirated and the wells were washed three times with phosphate-buffered saline. Next, the plates were air-dried overnight and stained with 0.1% crystal violet. The OD of the wells was measured at 600 nm using the ELISA reader device.

Disk Diffusion Method
MHA was used for antimicrobial susceptibility testing of the mentioned TiO 2 NPs. Anti-bacterial disks of the CMC-coated TiO 2 NP were evaluated for inhibition of microbial growth of the UTI-causing bacterial agents. In this step, concentrations from 1.568, 3.136, 6.272, and 12.544 mg/mL were used. The zone of bacterial growth inhibition was inspected after planting the discs on the Muller-Hinton media. Evaluating the zone of inhibition after 24 hours showed that the prepared nano-agent was effectively capable of suppressing the bacterial growth on all UTI-causing bacteria at all different TiO 2 concentrations. In Table 4, the diameter of the zone of inhibition on the bacterial species was represented in millimeters. The results of the disk diffusion method using standard antibiotic disks demonstrated that E. coli had the highest resistance to antibiotics ampicillin, while cephalothin and cotrimoxazole had the highest sensitivity to antibiotics gentamicin and amikacin. P. aeruginosa exhibited the highest resistance to antibiotics ciprofloxacin and the highest sensitivity to gentamicin. Klebsiella represented the highest resistance to antibiotics ampicillin and the highest sensitivity to antibiotics gentamicin and amikacin. S. pneumonia had the highest resistance to antibiotics penicillin and the highest sensitivity to antibiotics erythromycin. S. aureus developed the highest resistance to antibiotics ceftriaxone and cefixime, as well as the highest sensitivity to antibiotics cotrimoxazole. S. epidermis had the highest resistance to antibiotics oxacillin and the highest sensitivity to antibiotics cephalothin.

Characteristics of TiO 2 NPs
Details of this component are TiO 2 , anatase, average particle size of 10-25 nm, the specific surface area of 200-240 m 2 /g, pH rate of 6-6.5, white color, purity of > 99%, bulk density of 0.24 g/cm 3 , true density of 3.9 g/cm 3 , loss of weight in drying of 4.17%, and loss of weight on ignition equal to 8.24% (Figures 1-3).

Minimum Inhibitory Concentration Results
In this test, the MIC of nanoparticles was determined by the microdilution method for S. pneumoniae, S. aureus, S. epidermidis, E. coli, P. aeruginosa, K. pneumoniae, and C. albicans. Microbial growth rates in the presence of different concentrations of TiO 2 NPs were calculated in 600 nm OD (Tables 5-7). For MBC detection, two wells before and after the MIC well cultured on Mueller Hinton agar media.  (Table 8). Thus, TiO 2 NPs are effective antimicrobial agents for grampositive, Gram-negative bacteria, and C. albicans, but their inhibitory effect on yeast was greater than bacteria.

Minimum Biofilm Inhibitory Concentration
The results of biofilm inhibition assay using CMC coated     Tables 9-11, the MBIC of more than 90% of TiO 2 NPs was 6.25 mg/mL for both Gram-negative and Gram-positive bacterial types and 1.562 mg/mL for C. albicans.

Discussion
In recent years, physicians and microbial scientists have found that the causative agents of microbial infections are becoming resistant to common antimicrobial medicine.
On the other side, by the acceleration of science and a better understanding of new materials with the potential for treating infection, many have focused on studying the antimicrobial properties of these materials in order to find new substitutions for ordinary antibiotics (22). As previously stated, biofilms are a major cause of infectious diseases and could increase the tolerance of bacteria inside them toward different antibiotics (23). Therefore, finding a drug that could penetrate into the biofilm and destroy them, would be helpful for effectively battling infections. Inhibition of biofilm-associated diseases requires incredible attention since this structure has high levels of resistance to antibiotics (24). Several studies recommended combination therapy as the treatment of choice in biofilmassociated infections while considering macrolides as one of the first selected antibiotics, but many of these microbial agents of biofilm construction have become resistant to them over time. Biofilms can be up to multiple-fold more resistant to antimicrobial agents compared to planktonic forms due to various mechanisms (25). The current time could be considered as the age of nanotechnology for its fascinating applications and encouraging results of their use for improving different aspects of human life. An understanding of NPs affecting the critical causative agent of microbial infections allows us to gain a mechanistic understanding of toxicity and guides us toward designing rules for creating safe antimicrobial nanomaterials (26). As mentioned earlier, an NP is a small-sized particle with a dimension of less than 100 nm. NP studies are a field of intensive research interest due to a broad diversity of potential applications in electronic, biomedical, and optical fields. These particles have the capacity to attach and penetrate into bacterial cells, disrupt the bacterial membrane, and interact with chromosomal DNA (27). The purpose of this research was to provide new insights about using CMC-coated photocatalytic TiO 2 NPs as antimicrobial agents against different gram-positive, gram-negative bacteria, and C. albicans. In this study, the antimicrobial effect of CMCcoated photocatalytic TiO 2 NPs was investigated on seven common agents of many infections, including S. pneumoniae, S. aureus, S. epidermidis, E. coli, P. aeruginosa, K. pneumoniae, and C. albicans. The antibacterial effects of these materials were evaluated using the microdilution method. The biofilm inhibition effects of TiO 2 -coated NPs were determined as well. The results of our test indicated that strong biofilms will form on the plates in the absence of the TiO 2 NPs, but adding the particles would strongly inhibit biofilm formation and maturation. The result showed that the MICs of TiO 2 NPs for E. coli, P. aeruginosa, and K. pneumoniae, as the Gram-negative bacteria, were 1.489, 1.208, and 1.166 mg/mL, respectively. Further, these values were 0.512, 0.830, and 0.707 mg/mL for S. pneumoniae, S. aureus, and S. epidermidis as the Gram-positive bacteria, respectively. Moreover, it was equal to 0.253 mg/mL for C. albicans as the yeast strain. The MBIC of more than 90% of TiO 2 NPs was 6.25 mg/ mL for both Gram-negative and Gram-positive bacterial types and 1.562 mg/mL for C. albicans. In their study, Jalvo et al focused on measuring the antibiofilm and antimicrobial properties of functionalized surfaces coated by photocatalytic TiO 2 NPs against Pseudomonas putida and S. aureus. They presented that the antimicrobial activity of TiO 2 led to wide cover damage. The decrease of cell inviability was more than 99.9% for TiO 2 NPs on the glass level (21). In another study, Verdier et al examined the antimicrobial effect of photocatalytic TiO 2 alone or in coatings on E. coli bacterium and measured various types of parameters that significantly influence the antimicrobial activity (28). Similarly, Lavaee et al reported the results of investigating the antibacterial and biofilm inhibition activity of silver, TiO 2 , and iron NPs. They aimed at comparing the antibacterial effects of erythromycin, clindamycin, chlorhexidine, penicillin, vancomycin, and tetracycline with TiO 2 , iron, and Ag NPs and consider the synergistic antimicrobial and biofilm inhibition effects of these NPs in pathogenic and standard species of Streptococcus sanguinis and Streptococcus mutans. Based on their findings, the use of NPs alone showed a higher MIC than using them synergistically. The most efficient synergistic resolving agent was the one containing Ag, TiO 2 , and Fe 3 O 4 (29). In another study, Vincent et al evaluated the efficacy of TiO 2 and zinc oxide nanomaterials against biofilm-producing and metallo beta-lactamase P. aeruginosa aiming at determining the effect of TiO 2 and zinc oxide NPs on biofilm-producing and metallo beta-lactamase P. aeruginosa. These nanomaterials demonstrated perceptible activity at whole experimental concentrations, and it was revealed that TiO 2 and zinc oxide nanomaterials may serve as promising antimicrobial agents in this regard (30).

Conclusions
Overall, the results of the current study demonstrated that TiO 2 NPs are effective antimicrobial agents for E. coli, P. aeruginosa, and K. pneumoniae, as Gram-negative bacteria, as well as S. pneumoniae, S. aureus, and S. epidermidis, as Gram-positive bacteria, and C. albicans, as the yeast strain, and repressed biofilm composition for the first time. In addition, the findings confirmed that the efficiency for the inhibition of both microbial and fungi biofilms is of great importance for these particles and they could be used against a diversity of infections. Although the results of the present study seem to be successful, further studies are required so that to consider these agents for clinical applications.