Research on the effect of TiO2 nanotubes coated by gallium nitrate on Staphylococcus aureus‐Escherichia coli biofilm formation

Abstract Background In clinical practice, the cases with bacterial infection caused by titanium implants and bacterial biofilm formation on the surface of titanium materials implanted into human body can often be observed. Thus, this study aimed to demonstrate whether the mixed biofilm of Staphylococcus aureus/Escherichia coli can be formed on the surface of titanium material through in vitro experiments and its formation rules. Methods The titanium plates were put into the well containing S aureus or/and E coli. Bacterial adhesion and biofilm formation were analyzed by crystal violet, XTT method, confocal laser scanning microscopy, and scanning electron microscopy. Results The results of bacterial adhesion in each group at 6‐72 hours showed that the number of bacterial adhesion in each group was increased with the extension of time and reached to the highest level at 72 hours. Moreover, the biofilm structure in the S aureus‐E coli group was significantly more complex than that of the simple S aureus group or E coli group, and the number of bacteria was also significantly increased in the S aureus‐E coli group. Conclusion Those data provide a laboratory basis for the prevention and treatment of mixed infection of subsequent biological materials.

biomechanical properties and histocompatibility than stainless steel and other materials in the past. At present, titanium and its alloy materials are the most commonly used materials in orthopedics and stomatology.
It is of great clinical significance to study the infection caused by its implantation from the most commonly used materials. However, due to the fact that titanium alloy materials are composed of a variety of elements and there are many interference and confounding factors, it is difficult to carry out the subsequent nano-modification for titanium alloy materials. Therefore, pure titanium metal was selected as the material for the in vitro study in this experiment, so as to provide ideas and laboratory basis for the subsequent study of titanium alloy and nickel-cobalt alloy.
With the abuse of antibiotics, the emergence of drug-resistant strains and the high incidence of some immunodeficiency diseases, mixed infection has become a difficult problem in clinical work, especially for the diseases in intensive care unit such as infections caused by various catheter implantation, orthopedic diabetic foot, and chronic osteomyelitis caused by internal fixation implantation.
Houshian et al reviewed and studied 418 patients with hand surgical infection, with a mixed infection rate of 11.7%. 3 A large number of studies at home and abroad have shown that the mixed bacterial biofilm formed on the surface of the material after the mixed bacterial infection has more complex morphology and biological characteristics. 4 However, the related research on the mixed biofilm of Staphylococcus aureus and Escherichia coli on the surface of titanium metal material has not been reported at home and abroad. Therefore, in this study, titanium plates were mixed with S aureus-E coli to observe whether mixed biofilms could be formed on the surface of titanium and the formation rules and characteristics. In addition, a control study was conducted with a single bacterial biofilm to explore the causes of complex, severe, and refractory mixed infection, hoping to provide a laboratory basis for the prevention and treatment of mixed infection of subsequent biological materials.

| Preparation of titanium plate materials
The pure titanium plates with 5 × 5 mm in size are selected, polished with sandpaper in order of 800 mesh, 1000 mesh, 1200 mesh, and 2000 mesh, washed with deionized water for 15 minutes, and immersed in HF + HNO 3 + H 2 O = 1:1:8 solution for 15 seconds to carry out chemical polishing. After that, deionized water was added to stop the reaction and washed for 15 minutes. Then, titanium plates were taken out and put into steam autoclave sterilizer, with a pressure of 103.4 kPa (1.05 kg/ cm 2 ) and a temperature of 121.3°C, for 30 minutes, and then taken out and dried in the drying oven for 30 minutes, and placed for standby.

| Bacterial culture
Staphylococcus aureus and E coli were purchased from American Type Culture Collection (ATCC). The standard strains of S aureus (ATCC 25923) and E coli (ATCC 25922) were inoculated to MH agar plate and cultured for 24 hours at 37°C. Subsequently, 12 mL tryptic soy broth (TSB) culture medium was put into the test tube. The inoculation ring was used to select a single colony on the bacteria plate of each group (repeat for three times) and gently vibrate the liquid level junction in the test tube. The bacteria in each group were inoculated into the tube containing 12 mL TSB medium, and the tube was placed in a constant humidity oscillator at 37°C and 150 r/min for 16-18 hours. After the growth of bacterial cells to the logarithmic growth stage, the concentration of bacterial solution in each group was adjusted to 1 × 10 7 CFU/mL for later use with ultraviolet spectrophotometer and TSB medium. The mixed bacterial solution from mixed group was prepared according to 1:1, such as 2 mL mixed bacterial solution (1 mL S aureus bacterial solution + 1 mL E coli bacterial solution).

| Experimental grouping
The experiment was divided into four groups: TSB-treated control group (Group A), E coli-treated group (Group B), S aureus-treated group (Group C), and E coli and S aureus mixture-treated group (Group D). 4 sterile 24-well culture plates were taken, and three wells were selected in each plate and added 2 mL 1 × 10 7 CFU/mL prepared bacterial solution (TSB for control group, TSB containing S aureus, TSB containing E coli, and TSB containing S aureus + E coli for experimental groups), the prepared sterilized titanium plates with the size of 5 mm × 5 mm were put into the well containing bacterial solution, and titanium plates were immersed into the bacterial solution with sterilizing tweezers and cultured in a 37°C incubator. After cultured for 6, 12, 24, 48, and 72 hours, titanium plates were taken out for bacterial biofilm determination and analysis.

| Semiquantitative detection of bacterial adhesion and biofilm formation by crystal violet (CV)
After cultured for 6, 12, 24, 48, and 72 hours, 100 μL bacterial solution was drawn from the corresponding wells, respectively. The absorbance was measured at 620 nm with a multifunctional microplate reader and the data was recorded for standby. The corresponding titanium plates were taken out, transferred to the new 24 well plate, and added 2.5 mL PBS to rinse twice to remove the floating bacteria on the metal plate. After gently washing the bacteria and discarding PBS, the titanium plates were transferred to the absorbent paper, put into the clean 24-well plate after air drying, added 400 μL 2% CV dye solution to dye each well, and incubated at 37°C for 30 minutes. Then, CV dye solution was sucked and discarded, and 2.5 mL PBS was added to rinse for 3 times. The titanium plate was transferred to the absorbent paper, dried at room temperature, and transferred to a new 24 well plate. After adding 400 μL DMSO to decolorize for 15 minutes, 100 μL decolorizing solution was taken from each well to 96 well plate. The optical density (OD) value of 570 nm absorbance was measured by microplate reader.
The statistical chart of bacterial adhesion at different time points and groups was made with the culture time as X axis and 570 nm absorbance as Y axis. The average OD of the TSB-treated control well was subtracted from the measured value of the well, and the measured value of the OD was corrected. Formula biofilm formation (BF) = (AB − CW)/G was used to evaluate the ability of biofilm formation. AB is the OD value of bacteria at the 570 nm, CW is the OD value of TSB-treated control group at 570 nm, and G is the OD value of bacterial solution at the 620 nm. 5,6 BF ≥ 1.1 represents strong biofilm, BF between 0.7 and 1.09 represents mature biofilm, BF between 0.35 and 0.69 represents weak biofilm, and BF < 0.35 represents no biofilm formation. After 6,12,24,48, and 72 hours of culture, the titanium plates at the corresponding time points were taken out, transferred to the new 24 well plate, and gently washed twice with 4°C sterile 2.5 mL PBS to remove the floating bacteria on the metal plate. After discarding PBS, the titanium plates were dried naturally and then put into the 24-well plate.

| Detection for the dynamic of bacterial biofilm formation by XTT method
Each well was added with 300 μL TSB culture medium and 60 μL XTT solution, and incubated in 37°C at dark for 2 hours. After the incubation, 120 μL medium was taken out from each well to measure the cell activity of the bacteria.

| Observation of bacterial biofilm formation thickness and capacity by confocal laser scanning microscopy (proportion of live and dead bacteria)
The titanium plates were taken out for 12, 24, 48, and 72 hours after culture and gently washed with 4°C sterile 2.5 mL PBS twice to remove the floating bacteria on the metal plate. Then, 400 μL fluorescent dye solution (SYTO9 and PI dyes for staining living bacteria and dead bacteria, respectively) was added and dyed at room temperature for 15 minutes. After that, the titanium plates were taken out and put into 2.5 mL physiological saline for light rinsing. After absorbing the excess fluorescent dye, the titanium plates were put on the slide to fluorescently imaged under the confocal laser scanning microscopy (CLSM).
Observation conditions: The argon ion laser is used as the light source.

| Ultrastructural observation of bacterial biofilm by scanning electron microscope (SEM)
The titanium plates were taken out for 6, 12, 24, 48, and 72 hours, washed with 2.5 mL PBS, and fixed in refrigerator at 4°C for 24 hours. Then, the plates were washed with PBS solution for 3 times and fixed with 1% starving acid solution at 4°C for 2 hours.
After that, the titanium plates were dehydrated with ethanol gradient for 20 minutes, replaced with isoamyl acetate for 20 minutes, and frozen at −20°C following permeating with tert-butyl alcohol at 40°C for 2 hours. The formation of biofilm on the surface of the specimen was observed under SEM.

| Statistical analysis
SPSS19 statistical software was used for analysis. The data are expressed as mean ± standard deviation. The pairwise comparison of multiple groups was performed by ANOVA. P values < .05 were considered statistically significant.

| Bacterial adhesion on titanium surface
The four groups were co-cultured with titanium plates for 6, 12,

| Results of ability of bacterial biofilm formation at different time points on the surface of titanium plate
As shown in Figure 2, no bacterial biofilm was formed in E coli group, S aureus group, and mixed group at 6 hours; no bacterial biofilm was formed in E coli group at 12 hours, but weak bacterial biofilm was formed in S aureus group and mixed group at 12 hours, and there was no significant difference between the two groups (P > .05); weak bacterial biofilm was formed in E coli group and S aureus group at 24 hours, mature bacterial biofilm was formed in mixed group at 24 hours, and it was found that there was a statistically significant difference between the E coli group and the mixed group (P < .05), while there was no statistically significant difference in other groups (P > .05). At 48 hours, weak bacterial biofilm was formed in E coli group and S aureus group, and mature bacterial biofilm was formed in mixed group, which was statistically significant compared with E coli group and S aureus group (P < .05). At 72 hours, mature bacterial biofilm was formed in E coli group, S aureus group, and mixed group, which was statistically different between E coli group and S aureus as well as mixed group (P < .05), and there was no statistical significance between S aureus group and mixed group (P > .05).

| Test results of the forming dynamic of titanium bacterial biofilm
The TSB-treated control group, E coli group, S aureus group, mixed group, and titanium plate were cultured for 6, 12, 24, 48, and 72 hours, and the dynamic detection results of biofilm formation by XTT method. The results showed that only the biofilm-forming dynamic in the mixed group at 12 hours > E coli group,> S aureus group and > control group. There was no significant difference between mixed group and S aureus group as well as E coli group at 6, 24, 48, and 72 hours, which indicated that there was no obvious advantage in forming dynamic of biofilm by mixing S aureus and E coli (Figure 3).

| Detection results of bacterial biofilm on titanium surface by confocal laser scanning microscope (CLSM)
After forming bacterial biofilm on titanium surface at different time point, SYTO9 (green fluorescence) and PI (red fluorescence) dyes were used to stain living bacteria and dead bacteria, respectively.
As shown in Figure 4A, there were several punctate green and red fluorescence signals in the TSB-treated control group, but no bacterial biofilm formation. The formation of mature bacterial biofilm was observed in the E coli group; a large area of green fluorescence signal was observed. Similarly, the formation of mature bacterial biofilm was observed in the S aureus group; a large area of green fluorescence signal with dense lamellar arrangement can be seen. Compared with the two groups, the thickness of mixed group was greater than that of E coli group, but showed no statistical differences when compared with S aureus group. At 24 hours, the biofilm gradually thickened, and the thickness of the mixed group F I G U R E 3 Results of the forming dynamic of titanium bacterial biofilm in each group. The bacteria activity in each group at each time point was measured by XTT cell proliferation assay (*P < .05; **P < .01; ***P < .001) F I G U R E 4 CLSM observation of bacterial biofilms of each group at each time point. The SYTO9 (green fluorescence) and PI (red fluorescence) dyes were used to stain living bacteria and dead bacteria, respectively. A, Representative CLSM images of bacterial biofilms in each group at each point. B, Living bacteria ratio of bacterial biofilms of each group at 12-72 h. C, Dead bacteria ratio of bacterial biofilms of each group at 12-72 h was greater than that of E coli group, and there were no statistical differences between mixed and S aureus groups. At 48 hours, the thickness of biofilm increased slowly, and the thickness of the mixed group was larger than that of S aureus group and E coli group, which showed a significant difference. At 72 hours, the thickness of the biofilm was the thickest, and the thickness of the mixed group was larger than that of S aureus group and E coli group, which showed a significant difference ( Figure 5A). The three-dimensional reconstruction showed that during the co-culture period of 12-72 hours,

| Observation results of bacterial biofilm on titanium surface by Scanning electron microscopy (SEM)
Mature biofilm was observed in the mixed group, S aureus group, and E coli group. In the mixed group, it can be observed that the stacked masses formed by S aureus and E coli grew together. The biofilm structure in the mixed group was significantly more complex than that of the simple S aureus and E coli group, and the number of bacteria was also significantly increased ( Figure 6).

| D ISCUSS I ON
In this paper, pure titanium was used as the carrier of biomate-  there was no significant difference in the amount of biofilm, compared with the single culture of E faecium, and the amount of mixed biofilm was higher than that of E faecium.
It is clear that S aureus and E coli can form mixed biofilm on the surface of titanium plate, but the following problem is the relationship between the two strains in the mixed biofilm. At the same time, the formation of single bacterial biofilm and mixed biofilm was studied. The results showed that the number of S aureus/E coli mixed biofilm was more than that of single biofilm at each time point, but the forming dynamic (bacterial activity) was stronger than that of single biofilm only at 12 hours, there was no difference between single biofilm at 24 and 48 hours, and the dynamic of mixed biofilm at 72 hours was less than that of single biofilm. At the same time, the CLSM results also showed that the number of mixed biofilm increased, but the proportion of dead bacteria increased, and the activity of biofilm decreased. SEM observation showed that there were not the same number of two strains in the mixed biofilm, but mainly E coli. The above results indicate that after the formation of mixed organisms, their activity is inhibited and the majority of them are E coli. Maybe, there is a competitive relationship between S aureus and E coli, which makes the E coli gain the competitive advantage. amount of bacteria than that of S aureus, indicating that the time for the formation of E coli is shorter and the number is more, which may enable it to develop and maintain advantages in the mixed biofilm. In addition, S aureus in mixed biofilms may also provide some adhesion factors that E coli does not have, so that E coli can maintain dominant growth in mixed biofilms. These factors may be the reason why E coli keeps the advantage in the mixed biofilm and S aureus is inhibited in the mixed infection. In order to verify the relationship between the two, the future experiments can separate and quantitatively analyze S aureus and E coli in the mixed bacterial biofilm, and further explore the mechanism and role of their competition.
Christensen et al 5 first described the quantitative analysis of bacterial biofilm with CV staining in 1985, then improved its accuracy and modified it, and made the biofilm quantified in the well plate. 19 CV is an alkaline dye that binds to negatively charged surface molecules and polysaccharides in the extracellular matrix. 20 Because bacteria, whether living or dead, and the substrate are stained with CV, it is not suitable to evaluate the activity of biofilm. 21 In this experiment, CV method was used to detect the total amount of bacterial biofilm, and the results showed that the total amount of mixed biofilm was larger than that of single biofilm, but it contained dead bacteria, which could not reflect the activity of the overall bacterial biofilm in real time. In order to distinguish living bacteria from dead ones, detect and quantify the metabolic activity in living bacteria, the use of various reactive dyes has become a feasible technology and method, including 5-cyano-2,3-ditolyl tetrazolium chloride, and XTT. 22,23 XTT method is based on the reduction of XTT dye to water-soluble methionine. 24 The absorbance of bacterial supernatant is in direct proportion to the number of metabolic active microbial cells. XTT method has been widely used in quantitative analysis of cells and bacteria in planktonic culture. The results of this experiment also showed that although the amount of mixed bacterial biofilm measured by CV staining method was larger than that of single biofilm, XTT found that there was no difference between mixed biofilm and single biofilm at 24 and 48 hours, but the dynamic of mixed biofilm at 72 hours was smaller than that of single biofilm, which indicated that although the total amount of mixed bacterial biofilm was large, the number of active bacteria in mixed biofilm was reduced.
CLSM is a tool widely used in biofilm observation, because it can obtain three-dimensional images of biofilm structure and monitor its development over time. The fluorescent dyes SYTO9 and PI are a kind of nucleic acid dye. SYTO9 makes the living bacteria emit green fluorescence, while PI makes the dead bacteria emit red fluorescence. It diffuses passively through the cell membrane and binds with the DNA of the living and dead cells. 25 Since DNA is also an important component of extracellular matrix, 26 this staining will provide information about the total biofilm biomass. SYTO9 was used to study the composition and morphology of biofilm by CLSM. 27 The dye is also used for conventional quantification of bacterial and yeast biofilm biomass. 28,29 Compared with single biofilm at the same time point, the thickness of mixed biofilm is thicker, the morphology is more complex, and the proportion of dead bacteria in mixed biofilm is increased, and the number of living bacteria is reduced and the number of dead bacteria is increased with the extension of culture time. SEM can show the detailed surface morphology of microbial biofilm and its structure. The SEM images of this experiment confirmed the CLSM analysis. The morphology of the mixed biofilm of S aureus and E coli is more complex than that of a single biofilm.
The biofilm is composed of rod-like clusters and layered. The number is less, and the morphology is more complex and dense with the increase of culture time.
In conclusion, quantitative and qualitative analysis methods were used to observe the formation of S aureus-E coli mixed biofilm, and the rules of its formation were summarized and discussed in these experiments. Compared with single bacterial biofilm, the number and shape of mixed biofilm were more complex, and changed with the change of culture time. The formation process of mixed biofilm is closely related to the interrelationship between the two bacteria.
Why they form complex mixed biofilm and what molecular mechanism regulates their formation process, which will be studied in the future.